Methods and compositions for the modulation of cell interactions and signaling in the tumor microenvironment

ABSTRACT

The present invention is generally directed to identify interacting cells in the tumor microenvironment and using the identified interactions to enhance anti-tumor immunity in cancer. Identified interactions can be modulated using therapeutic agents. Immune cells resistant to suppression can be used for adoptive cell transfer. The present invention is also generally directed to cell types and genes that are correlated to time of tumor growth and tumor size.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/910,340, filed Oct. 3, 2019. The entire contents of theabove-identified application are hereby fully incorporated herein byreference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (“BROD-4320US_ST25.txt;”Size is 11,638 bytes (12 KB on disk) and it was created on Oct. 5, 2020)is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The subject matter disclosed herein is generally directed to targetinginteractions and signaling in the tumor microenvironment to enhanceanti-tumor immunity in the treatment of cancer.

BACKGROUND

Tumor cellular diversity poses both challenges and opportunities forcancer therapy. Various non-malignant cells comprise the tumormicroenvironment. The composition of the microenvironment has animportant impact on tumorigenesis and in the modulation of treatmentresponses. Interactions between cells play crucial roles in the tumormicroenvironment. The next wave of therapeutic advances in cancer willlikely be accelerated by emerging technologies that systematicallyassess the malignant, microenvironmental, and immunologic states mostlikely to inform treatment response and resistance. New tools, such assingle-cell genomics, have allowed for mapping single cell types in atissue. A comprehensive cell atlas makes it possible to catalog all celltypes and even subtypes of cells in a tissue, and even distinguishdifferent stages of differentiation and cell states, such as immune cellactivation. There is a need to further understand communication betweencell types during tumor progression.

SUMMARY

In one aspect, the present invention provides for an isolated CD8 T cellmodified to decrease or eliminate expression, activity or function ofNRP1.

In another aspect, the present invention provides for an isolated CD8 Tcell modulated to decrease or eliminate expression, activity or functionof CRTAM.

In certain embodiments, the T cells express a chimeric antigen receptor(CAR) or endogenous T cell receptor (TCR). In certain embodiments, the Tcells are tumor infiltrating lymphocytes (TILs).

In another aspect, the present invention provides for a method ofenhancing an anti-tumor immune response in a subject in need thereofcomprising administering to the subject an antagonist of VEGFB-NRP1interaction. In certain embodiments, the antagonist is an antibody orfragment thereof specific for NRP1. In certain embodiments, theantagonist is an antibody or fragment thereof specific for VEGFB.

In another aspect, the present invention provides for a method ofenhancing an anti-tumor immune response in a subject in need thereofcomprising administering to the subject an antagonist of CADM1-CRTAMinteraction. In certain embodiments, the antagonist is an antibody orfragment thereof specific for CADM1. In certain embodiments, theantagonist is an antibody or fragment thereof specific for CRTAM.

In another aspect, the present invention provides for a method ofenhancing an anti-tumor immune response in a subject in need thereofcomprising administering to the subject an antagonist of TFPI-SDC4interaction. In certain embodiments, the antagonist is an antibody orfragment thereof specific for TFPI. In certain embodiments, theantagonist is an antibody or fragment thereof specific for SDC4.

In another aspect, the present invention provides for a method ofenhancing an anti-tumor immune response in a subject in need thereofcomprising administering to the subject an antagonist of MFGE8-ITGAVinteraction. In certain embodiments, the antagonist is an antibody orfragment thereof specific for MFGE8. In certain embodiments, theantagonist is an antibody or fragment thereof specific for ITGAV.

In another aspect, the present invention provides for a method ofenhancing an anti-tumor immune response in a subject in need thereofcomprising administering to the subject an antagonist of ADAM12-SDC4interaction. In certain embodiments, the antagonist is an antibody orfragment thereof specific for ADAM12. In certain embodiments, theantagonist is an antibody or fragment thereof specific for SDC4.

In another aspect, the present invention provides for a method ofenhancing an anti-tumor immune response in a subject in need thereofcomprising administering to the subject an antagonist of CYR61-ITGAVinteraction. In certain embodiments, the antagonist is an antibody orfragment thereof specific for CYR61. In certain embodiments, theantagonist is an antibody or fragment thereof specific for ITGAV.

In another aspect, the present invention provides for a method ofenhancing an anti-tumor immune response in a subject in need thereofcomprising administering to the subject an antagonist of the interactionbetween FN1 with FBLN1 and/or ITGB1. In certain embodiments, theantagonist is an antibody or fragment thereof specific for FN1. Incertain embodiments, the antagonist is an antibody or fragment thereofspecific for FBLN1. In certain embodiments, the antagonist is anantibody or fragment thereof specific for ITGB1.

In another aspect, the present invention provides for a method ofenhancing an anti-tumor immune response in a subject in need thereofcomprising administering to the subject an antagonist of one or moreligand-receptor pairs in Table 1.

In certain embodiments, the antagonist is a small molecule, aptamer, orprotein capable of binding to a receptor or ligand. In certainembodiments, the antagonist is administered directly to the tumor. Incertain embodiments, the antagonist is administered intravenously orintraperitoneally to the subject. In certain embodiments, the antagonistis administered in a successive, sequential or simultaneous treatmentregimen.

In certain embodiments, the subject is administered an additionalimmunotherapy. In certain embodiments, the immunotherapy comprisesanti-PD-1, anti-CTLA4, anti-PD-L1, anti-TIM3, anti-TIGIT, anti-LAG3, orcombinations thereof.

In another aspect, the present invention provides for a method ofgenerating T cells for adoptive cell transfer to a subject sufferingfrom cancer comprising obtaining T cells; and modifying the T cells todecrease or eliminate expression, activity or function of one or moregenes selected from the group consisting of NRP1 and CRTAM.

In certain embodiments, the T cells are modified with a geneticmodifying agent capable of targeting the gene or gene transcript. Incertain embodiments, the genetic modifying agent comprises a CRISPRsystem, RNAi system, a zinc finger nuclease system, a TALE system, or ameganuclease. In certain embodiments, the CRISPR system comprises aCRISPR-Cas base editing system, a prime editor system, or a CAST system.

In certain embodiments, the T cells are CD8 T cells. In certainembodiments, the T cells are further modified to express a chimericantigen receptor (CAR) or exogenous T cell receptor (TCR). In certainembodiments, the T cells are obtained from a subject in need thereof. Incertain embodiments, the T cells are tumor infiltrating lymphocytes(TILs). In certain embodiments, the method comprises decreasing oreliminating expression, activity or function of NRP1. In certainembodiments, the method comprises decreasing or eliminating expression,activity or function of CRTAM. In certain embodiments, the methodfurther comprises eliminating expression, activity or function of one ormore additional genes selected from the group consisting of TRBC1, TRBC2and TRAC. In certain embodiments, the method further compriseseliminating expression, activity or function of one or more additionalgenes selected from the group consisting of checkpoint proteins. Incertain embodiments, the method further comprises eliminatingexpression, activity or function of one or more additional genesselected from the group consisting of HLA-A, HLA-B, HLA-C, and B2M.

In another aspect, the present invention provides for a method of cancertreatment comprising administering to a subject in need thereof the Tcells according to any embodiment herein or T cells generated accordingto any embodiment herein.

In another aspect, the present invention provides for a method ofdetermining tumors resistant to an immune response comprising detectingthe expression of CADM1 in tumor cells obtained from a subject in needthereof, wherein increased levels indicate resistant tumors.

In another aspect, the present invention provides for a method ofdetermining tumors resistant to an immune response comprising detectingthe expression of VEGFB in tumor cells obtained from a subject in needthereof, wherein increased levels indicate resistant tumors.

In another aspect, the present invention provides for a method ofdetermining tumors resistant to an immune response comprising detectingthe expression of TFPI in tumor cells obtained from a subject in needthereof, wherein increased levels indicate resistant tumors.

In another aspect, the present invention provides for a method ofdetermining tumors resistant to an immune response comprising detectingthe expression of MFGE8 in tumor cells obtained from a subject in needthereof, wherein increased levels indicate resistant tumors.

In another aspect, the present invention provides for a method ofdetermining tumors resistant to an immune response comprising detectingthe expression of ADAM12 in tumor cells obtained from a subject in needthereof, wherein increased levels indicate resistant tumors.

In another aspect, the present invention provides for a method ofdetermining tumors resistant to an immune response comprising detectingthe expression of FBLN1 in tumor cells obtained from a subject in needthereof, wherein increased levels indicate resistant tumors.

In certain embodiments, the interaction between cell types disrupts theextracellular matrix (ECM) in the tumor microenvironment and initiateswound healing signaling. In certain embodiments, the agent is capable ofblocking or disrupting wound healing signaling to Tregs.

In certain embodiments, the cancer is selected from the group consistingof melanoma, renal cancer, glioma, thyroid cancer, lung cancer, livercancer, pancreatic cancer, head and neck cancer, stomach cancer,colorectal cancer, urothelial cancer, prostate cancer, testicularcancer, breast cancer, cervical cancer, ovarian cancer and endometrialcancer.

These and other aspects, objects, features, and advantages of theexample embodiments will become apparent to those having ordinary skillin the art upon consideration of the following detailed description ofillustrated example embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the present inventionwill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of theinvention may be utilized, and the accompanying drawings of which:

FIGS. 1A-1D—Single cell clusters of B16 tumors. FIG. 1A. tSNE showingsingle cell clusters of the T cell component of B16 tumors. FIG. 1B.Projection of CD8 and CD4 expression on the T cell tSNE and pie chartsshowing the percentage of CD4 and CD8 T cells in each cluster. FIG. 1C.tSNE showing single cell clusters of the non-T cell component of B16tumors. FIG. 1D. Projection of the indicated genes on the non-T celltSNE.

FIG. 2—Diagram showing the methodology for determining interactionsbetween clusters.

FIG. 3—Heat maps showing the general upregulated ligands and receptorsin each cluster.

FIG. 4—Heat maps showing the specific upregulated ligands and receptorsin each cluster.

FIG. 5—Table showing interactions between ligand receptor pairs. The 1cluster indicates the cell clusters that the left gene in the pair isexpressed in and r_cluster indicates the cell clusters that the rightgene in the pair is expressed in. The clusters indicate the cell type,for example, T_X2 Treg; M_X4,6,3 Macrophages; T_X1,4,5,6,9,10 CD8 Tcells; M_X9,11,14 malignant cells (see also, FIG. 1A-D).

FIGS. 6A-6C—FIG. 6A. Projection of the indicated genes on the T cell andnon-T cell tSNE. FIG. 6B. Projection of the indicated genes on the Tcell and non-T cell tSNE. FIG. 6C. Projection of the indicated genes onthe T cell and non-T cell tSNE.

FIG. 7—Heat maps showing interacting clusters that express generalupregulated ligands and receptors pairs.

FIG. 8—Heat maps showing interacting clusters that express specificupregulated ligands and receptors pairs.

FIG. 9—Network plots showing specific upregulation (30%) interactionsfor clusters using all ligand-receptor pairs.

FIG. 10—Network plots showing specific upregulation (30%) interactionsfor clusters using ligand-receptor pairs with a T cell component.

FIGS. 11A-11B—FIG. 11A. Ligand-receptor network. FIG. 11B. Zoom in ofindicated ligand-receptor networks.

FIGS. 12A-12B—FIG. 12A. Ligand-receptor network for ITGAV and projectionon the tSNE plots. FIG. 12B. Diagram of interacting cells through ITGAV.

FIGS. 13A-13B—FIG. 13A. Ligand-receptor network for SDC4 and projectionon the tSNE plots. FIG. 13B. Diagram of interacting cells through SDC4.

FIG. 14—Interaction between ligand-receptor networks for SDC4 and ITGAV.

FIG. 15—Diagram showing the tumor microenvironment.

FIG. 16—Validations of interacting cells using immunohistochemistry.

FIG. 17—Validations of interacting cells using immunohistochemistry.

FIG. 18—Validations of interacting cells using immunohistochemistry.

FIG. 19—Expression of VEGFB in different human tumor types from thecancer genome atlas (TCGA).

FIGS. 20A-20C—FIG. 20A. Projection of the indicated genes on the T celland non-T cell tSNE. FIG. 20B. Expression of FBLN1 in melanoma patientsand the overall survival for patients having high or low expression.FIG. 20C. String data showing interactions between ligands andreceptors.

FIG. 21—Graph showing sample information for mice used in the study.Each point represents a mouse at each time point and the size of thetumor in the mouse.

FIG. 22—The number of single T cells and non-T cells from each sample.

FIGS. 23A-23B—FIG. 23A. UMAP analysis showing 9 annotated T cellclusters. FIG. 23B. Expression of cell type markers projected on theUMAP plot.

FIGS. 24A-24B—FIG. 24A. Heat map showing T cell cluster marker genes.FIG. 24B. Graph showing T cell cluster marker genes.

FIG. 25—UMAP analysis showing 12 annotated non-T cell clusters.

FIGS. 26A-26B—FIG. 26A. Heat map showing non-T cell cluster markergenes. FIG. 26B. Graph showing non-T cell cluster marker genes.

FIG. 27—Graph showing the number of up-regulated ligands or receptors inthe T cell and non-T cell clusters.

FIG. 28—Graphs showing GSEA for ligand/receptor up-regulation. Pointsabove the line are enriched upregulated for ligand or receptors.

FIG. 29—Heat map showing cluster similarity and the correlation ofligand/receptor average expression profile.

FIGS. 30A-30D—FIG. 30A. Cluster network of interactions between T andnon-T cells based on ligand and receptor pairs. FIG. 30B. Clusternetwork of interactions between T and non-T cell clusters. FIG. 30C.Cluster network of interactions among T cell clusters. FIG. 30D. Clusternetwork of interactions among non-T cell clusters.

FIG. 31—Gene network. All ligand-receptor pairs that are up-regulated inclusters. Node colored by type of interactions the gene is involved.

FIG. 32—Gene network. Ligand-receptor pairs that are up-regulated inclusters. Only genes and interactions between T and non-T cell clustersare shown. Node colored by T cell cluster the interactions are. Nodeborder colored by if the gene is up-regulated in T, M (non-T) or bothtypes of cell clusters.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS General Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Definitions of common termsand techniques in molecular biology may be found in Molecular Cloning: ALaboratory Manual, 2^(nd) edition (1989) (Sambrook, Fritsch, andManiatis); Molecular Cloning: A Laboratory Manual, 4^(th) edition (2012)(Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (AcademicPress, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B.D. Hames, and G. R. Taylor eds.): Antibodies, A Laboratory Manual (1988)(Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2^(nd) edition2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney,ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008(ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829);Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology andMolecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed.,John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Janvan Deursen, Transgenic Mouse Methods and Protocols, 2^(nd) edition(2011) .

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The term “optional” or “optionally” means that the subsequent describedevent, circumstance or substituent may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The terms “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, are meant to encompass variations of and from thespecified value, such as variations of +/−10% or less, +1-5% or less,+/−1% or less, and +/−0.1% or less of and from the specified value,insofar such variations are appropriate to perform in the disclosedinvention. It is to be understood that the value to which the modifier“about” or “approximately” refers is itself also specifically, andpreferably, disclosed.

As used herein, a “biological sample” may contain whole cells and/orlive cells and/or cell debris. The biological sample may contain (or bederived from) a “bodily fluid”. The present invention encompassesembodiments wherein the bodily fluid is selected from amniotic fluid,aqueous humour, vitreous humour, bile, blood serum, breast milk,cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph,perilymph, exudates, feces, female ejaculate, gastric acid, gastricjuice, lymph, mucus (including nasal drainage and phlegm), pericardialfluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skinoil), semen, sputum, synovial fluid, sweat, tears, urine, vaginalsecretion, vomit and mixtures of one or more thereof. Biological samplesinclude cell cultures, bodily fluids, cell cultures from bodily fluids.Bodily fluids may be obtained from a mammal organism, for example bypuncture, or other collecting or sampling procedures.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

Various embodiments are described hereinafter. It should be noted thatthe specific embodiments are not intended as an exhaustive descriptionor as a limitation to the broader aspects discussed herein. One aspectdescribed in conjunction with a particular embodiment is not necessarilylimited to that embodiment and can be practiced with any otherembodiment(s). Reference throughout this specification to “oneembodiment”, “an embodiment,” “an example embodiment,” means that aparticular feature, structure or characteristic described in connectionwith the embodiment is included in at least one embodiment of thepresent invention. Thus, appearances of the phrases “in one embodiment,”“in an embodiment,” or “an example embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment, but may. Furthermore, the particular features,structures or characteristics may be combined in any suitable manner, aswould be apparent to a person skilled in the art from this disclosure,in one or more embodiments. Furthermore, while some embodimentsdescribed herein include some but not other features included in otherembodiments, combinations of features of different embodiments are meantto be within the scope of the invention. For example, in the appendedclaims, any of the claimed embodiments can be used in any combination.

Reference is made to PCT/US2018/053791, PCT/US2018/061812,PCT/US2017/050469, PCT/US2016/059507, PCT/US2016/059487 andPCT/US2016/059463.

All publications, published patent documents, and patent applicationscited herein are hereby incorporated by reference to the same extent asthough each individual publication, published patent document, or patentapplication was specifically and individually indicated as beingincorporated by reference.

Overview

The tumor microenvironment (TME) is the environment around a tumor,including the surrounding blood vessels, immune cells, fibroblasts,signaling molecules and the extracellular matrix (ECM). Theextracellular matrix (ECM) surrounds all tumors to create a tumormicroenvironment (TME). Tumors disrupt the ECM as tumors can growwithout the ECM. Disruption of the ECM activates wound healing resultingin a suppressive immune environment. Single cell data provides granularinformation about genes and the context in which they are expressedacross a range of cell types and can be used to analyze single cell typein the TME. Additionally, analyzing single cells across a time course ina tumor model can allow identification of cell types and genes that arepositively and negatively correlated with tumor progression over timeand tumor size. Cell types and genes that are associated (e.g.,positively and negatively correlated) with time and size (i.e., are upand down regulated with time and/or size) can be therapeutic anddiagnostic targets. As used herein “associated” and “correlated” referto genes and/or cell types that go up or down with a specific variable,such as time and size or other genes or cell types. Applicants used thetime course single cell analysis to identify networks of interactingcell types in the TME by comparing the expression of ligand andreceptors in the cell types. Interactions were identified by comparingthe expression of ligand receptor pairs between cells along the timecourse. Applicants have identified novel interactions between tumorcells and specific immune cell subtypes, between immune cells, between Tcells, between non-T cells, and between T cells and non-T cells. Theinteractions were further validated using immunohistochemistry. Theligand receptor pairs and single cell transcriptome data allows fornovel therapeutic targets for modulating the interactions. Applicantsidentified targets that mediate communication between immune cells andimmune cells with tumor cells. Interestingly, the targets includeproteins that interact with the ECM or are ECM proteins.

Overview of Interacting Cell Types in the TME

Disclosed herein is the identification of interacting cell types in atumor microenvironment, which may be used to generate modified CD8 Tcells, methods of enhancing anti-tumor immune response, and methods ofdetermining tumor resistance to immune response as disclosed furtherbelow. In certain embodiments, interacting cell types in the TMEinteract through ligand receptor interactions. In certain embodiments,the interactions result in a more favorable environment for tumorgrowth. In certain embodiments, interactions between integrins alter theECM resulting in would healing signaling, such as, ITGAV expressed onTregs and FN1. In certain embodiments, interactions between cell typessuppress or enhance anti-tumor immunity. In certain embodiments,interacting cells can be detected or modified. In certain embodiments,tumor cells interact with the tumor microenvironment to suppress ananti-tumor immune response. In certain embodiments, tumors activatesuppressive T cells (e.g., Tregs). In certain embodiments, tumorssuppress effector T cells. Applicants identified interactions betweentumor cells and immune cells, such as, VEGFB-NRP1 and CADM1-CRTAM. Incertain embodiments, receptor ligand pairs in Table 1 are detected ormodified. In certain embodiments, interactions are blocked or signalinginhibited by one or more agents to enhance anti-tumor immunity. Incertain embodiments, Treg interactions with other cell types are blockedor inhibited. In certain embodiments, interactions from T cells to non-Tcells are blocked or inhibited.

TABLE 1 Ligand-Receptor Interactions. T_X indicates T cell clusters(see, FIG. 23) and M_X indicates the non-T cell clusters (see, FIG. 25).For example T_X2 indicates the Treg cluster. mult.score. pair I_clustersr_clusters general XCL1_XCR1 T_X0:T_X4:T_X7:T_X9:M_X2 M_X10 Inf VIM_CD44T_X2:T_X6:T_X8:M_X1:M_X4:M_X5:M_X6:M_X T_X6:M_X1:M_X3:M_X5:M_X8:M_X98:M_X11 Inf VCAN_TLR2 M_X1:M_X6 M_X1:M_X5 Inf VCAN_SELL M_X1:M_X6T_X0:T_X3:M_X2:M_X3:M_X11 Inf VCAN_ITGB1 M_X1:M_X6T_X1:T_X5:T_X6:M_X2:M_X7 Inf VCAN_ITGA4 M_X1:M_X6 T_X1:M_X7:M_X9 InfVCAN_CD44 M_X1:M_X6 T_X6:M_X1:M_X3:M_X5:M_X8:M_X9 Inf TRF_TFRC M_X1:M_X5T_X4:T_X9:M_X3 Inf TNFSF9_TNFRSF9 M_X4:M_X7:M_X11T_X2:T_X7:T_X8:T_X9:M_X2:M_X6:M_X7 Inf TNFSF4_TNFRSF4 M_X7T_X2:T_X8:T_X9:M_X4:M_X6:M_X7 Inf TNFRSF9_TNFSF9T_X2:T_X7:T_X8:T_X9:M_X2:M_X6:M_X7 M_X4:M_X7:M_X11 Inf TNFRSF4_TNFSF4T_X2:T_X8:T_X9:M_X4:M_X6:M_X7 M_X7 Inf TLR2_VCAN M_X1:M_X5 M_X1:M_X6 InfTIMP2_ITGB1 T_X2:M_X3:M_X8 T_X1:T_X5:T_X6:M_X2:M_X7 Inf TIMP1_CD63 M_X4M_X7:M_X8 Inf THBS1_CD47 M_X1:M_X6:M_X8 T_X6:M_X3:M_X7:M_X11 InfSLPI_CD4 M_X1:M_X3:M_X5:M_X8 T_X2:T_X5:T_X6:M_X3 Inf SERPINE2_LRP1T_X4:T_X7 M_X1:M_X5:M_X6:M_X8:M_X9 Inf SELPLG_SELLT_X1:M_X2:M_X3:M_X7:M_X11 T_X0:T_X3:M_X2:M_X3:M_X11 Inf SELPLG_ITGB2T_X1:M_X2:M_X3:M_X7:M_X11 T_X7:M_X1:M_X9 Inf SELPLG_ITGAMT_X1:M_X2:M_X3:M_X7:M_X11 M_X1:M_X6:M_X8 Inf SELL_SELPLGT_X0:T_X3:M_X2:M_X3:M_X11 T_X1:M_X2:M_X3:M_X7:M_X11 Inf RTN4_NGFRM_X1:M_X7 M_X11 Inf RP519_C5AR1 T_X3:M_X0:M_X7:M_X12 M_X1:M_X6:M_X8:M_X9Inf QRFP_P2RY14 T_X0:M_X2 M_X3M_X10 Inf PSAP_LRP1 M_X1:M_X3:M_X5:M_X10M_X1:M_X5:M_X6:M_X8:M_X9 Inf PDCD1_PDCD1LG T_X4:T_X7:T_X9:M_X6:M_X7T_X2:T_X9:M_X7 2 Inf P2RY14_QRFP M_X3M_X10 T_X0:M_X2 Inf OSM_LIFRM_X1:M_X5:M_X6:M_X8 M_X3:M_X11 Inf LTB_TNERSF1A T_X2:T_X5:M_X0:M_X2:M_X6M_X1:M_X5:M_X9 Inf LTB_LTBR T_X2:T_X5:M_X0:M_X2:M_X6 M_X1:M_X5 InfLTB_CD40 T_X2:T_X5:M_X0:M_X2:M_X6 M_X1:M_X4:M_X7 Inf LRP1_SERPINE2M_X1:M_X5:M_X6:M_X8:M_X9 T_X4:T_X7 Inf LRP1_PSAPM_X1:M_X5:M_X6:M_X8:M_X9 M_X1:M_X3:M_X5:M_X10 Inf LIFR_OSM M_X3:M_X11M_X1:M_X5:M_X6:M_X8 Inf ITGB1_VCAN T_X1:T_X5:T_X6:M_X2:M_X7 M_X1:M_X6Inf ITGB1_TIMP2 T_X1:T_X5:T_X6:M_X2:M_X7 T_X2:M_X3:M_X8 Inf ITGA4_VCANT_X1:M_X7:M_X9 M_X1:M_X6 Inf IL1RN_IL1R2 M_X1:M_X6:M_X8T_X2:T_X7:T_X8:M_X4 Inf IL1R2_1L1RN T_X2:T_X7:T_X8:M_X4 M_X1:M_X6:M_X8Inf IL1B_IL1R2 M_X1:M_X4:M_X5:M_X6:M_X9 T_X2:T_X7:T_X8:M_X4 InfIL1B_ADRB2 M_X1:M_X4:M_X5:M_X6:M_X9 T_X3:M_X0:M_X9 Inf IL16_CD4M_X0:M_X2 T_X2:T_X5:T_X6:M_X3 Inf IL15_1L2RB M_X1:M_X7T_X0:T_X2:T_X7:M_X2 Inf IL10_1L10RB T_X2 T_X0:M_X2 Inf IL10_1L10RA T_X2T_X7 Inf IFNG_IFNGR2 T_X4:T_X7:T_X9:M_X2 T_X3:M_X1:M_X5 Inf IFNG_IFNGR1T_X4:T_X7:T_X9:M_X2 T_X0:T_X2:T_X5:M_X2 Inf ICOS_ICOSLT_X2:T_X5:T_X6:M_X6 M_X7 Inf HSP90B1_TLR7 T_X4:T_X8:T_X9:M_X3 M_X3M_X9Inf HSP90B1_TLR2 T_X4:T_X8:T_X9:M_X3 M_X1:M_X5 Inf HSP90B1_LRP1T_X4:T_X8:T_X9:M_X3 M_X1:M_X5:M_X6:M_X8:M_X9 Inf HP_ITGB2 M_X1:M_X5:M_X9T_X7:M_X1:M_X9 Inf HP_ITGAM M_X1:M_X5:M_X9 M_X1:M_X6:M_X8 Inf HMGB1_THBDT_X4:T_X8:M_X2:M_X3 M_X5 Inf HMGB1_SDC1 T_X4:T_X8:M_X2:M_X3 M_X8 InfH2-M3_KLRD1 M_X3 T_X0:T_X1:T_X4:T_X7:M_X2:MX3:MX4:M_X1 1 Inf H2-M3_KLRC1M_X3 T_X1:T_X4:T_X7:T_X9:M_X2 Inf H2-M3_CD4 M_X3 T_X2:T_X5:T_X6:M_X3 InfGNAI2_C5AR1 T_X2:T_X8:M_X4:M_X11 M_X1:M_X6:M_X8:M_X9 Inf FN1_ITGB7M_X1:M_X5:M_X6:M_X8 T_X2:T_X6:M_X4:M_X10 Inf FN1_CD79AM_X1:M_X5:M_X6:M_X8 M_X0:M_X12 Inf FN1_C5AR1 M_X1:M_X5:M_X6:M_X8M_X1:M_X6:M_X8:M_X9 Inf FASL_TNERSF1A T_X1:M_X2 M_X1:M_X5:M_X9 InfFASL_FAS T_X1:M_X2 M_X1:M_X7 Inf FAS_FASL M_X1:M_X7 T_X1:M_X2 InfF13A1_ITGB1 M_X1:M_X5:M_X6:M_X8 T_X1:T_X5:T_X6:M_X2:M_X7 Inf F13A1_ITGA4M_X1:M_X5:M_X6:M_X8 T_X1:M_X7:M_X9 Inf CXCL2_XCR1 M_X1:M_X6:M_X8:M_X9M_X10 Inf CXCL16_CXCR6 M_X4:M_X7:M_X8 T_X1:T_X4:T_X6:T_X7:M_X6 InfCSF1_CSF1R T_X7 M_X1:M_X5:M_X6:M_X9 Inf CDH5_CDH5 M_X3 M_X3 InfCDH1_KLRG1 M_X3 T_X2:T_X8 Inf CDH1_ITGB7 M_X3 T_X2:T_X6:M_X4:M_X10 InfCDH1_ITGAE M_X3 T_X2:M_X10 Inf CD86_CTLA4 M_X1:M_X4:M_X7:M_X8T_X2:T_X8:M_X6 Inf CD80_CTLA4 M_X7 T_X2:T_X8:M_X6 Inf CD80_CD28 M_X7T_X1:T_X5:T_X6:M_X2:M_X6 Inf CD63_TIMP1 MX7.MX8 M_X4 Inf CD47_THBS1T_X6:M_X3:M_X7:M_X11 M_X1.M_X6:M_X8 Inf CD44_VIMT_X6:M_X1:M_X3:M_X5:M_X8:M_X9 T_X2:T_X6:T_X8:M_X1:M_X4:M_X5:M_X6:M_X8:M_X11 Inf CD44_VCAN T_X6:M_X1:M_X3:M_X5:M_X8:M_X9 M_X1:M_X6 InfCD40_LTB M_X1:M_X4:M_X7 T_X2:T_X5:M_X0:M_X2:M_X6 Inf CD4_IL16T_X2:T_X5:T_X6:M_X3 M_X0:M_X2 Inf CD4_H2-DMA T_X2:T_X5:T_X6:M_X3M_X4:M_X10:M_X11 Inf CD28_CD86 T_X1:T_X5:T_X6:M_X2:M_X6M_X1:M_X4:M_X7:M_X8 Inf CD28_CD80 T_X1:T_X5:T_X6:M_X2:M_X6 M_X7 InfCD274_PDCD1 M_X7:M_X8 T_X4:T_X7:T_X9:M_X6:M_X7 Inf CD14_ITGB1M_X1:M_X5:M_X6:M_X8 T_X1:T_X5:T_X6:M_X2:M_X7 Inf CD14_ITGA4M_X1:M_X5:M_X6:M_X8 T_X1:M_X7:M_X9 Inf CCR5_CCL4 T_X2:M_X1:M_X6:M_X8T_X4:T_X7:T_X9:M_X2:M_X3 Inf CCL8_CCR2 M_X8T_X2:T_X6:M_X1:M_X2:M_X5:M_X6 Inf CCL8_CCR1 M_X8 T_X2:M_X1:M_X6:M_X8 InfCCL7_CXCR3 M_X1:M_X6:M_X8 T_X1:T_X2:T_X5:T_X6:M_X2:M_X3 Inf CCL7_CCR5M_X1:M_X6:M_X8 T_X2:M_X1:M_X6:M_X8 Inf CCL7_CCR2 M_X1:M_X6:M_X8T_X2:T_X6:M_X1:M_X2:M_X5:M_X6 Inf CCL7_CCR1 M_X1:M_X6:M_X8T_X2:M_X1:M_X6:M_X8 Inf CCL5_SDC4 T_X0:T_X1:M_X2:M_X7 T_X2:M_X1:M_X3 InfCCL5_SDC1 T_X0:T_X1:M_X2:M_X7 M_X8 Inf CCL5_CXCR3 T_X0:T_X1:M_X2:M_X7T_X1:T_X2:T_X5:T_X6:M_X2:M_X3 Inf CCL5_CCR5 T_X0:T_X1:M_X2:M_X7T_X2:M_X1:M_X6:M_X8 Inf CCL5_CCR1 T_X0:T_X1:M_X2:M_X7T_X2:M_X1:M_X6:M_X8 Inf CCL4_CCR8 T_X4:T_X7:T_X9:M_X2:M_X3 T_X2 InfCCL4_CCR5 T_X4:T_X7:T_X9:M_X2:M_X3 T_X2:M_X1:M_X6:M_X8 Inf CCL4_CCR1T_X4:T_X7:T_X9:M_X2:M_X3 T_X2:M_X1:M_X6:M_X8 Inf CCL3_CCR5T_X4:T_X7:T_X9:M_X1:M_X2:M_X8:M_X9 T_X2:M_X1:M_X6:M_X8 Inf CCL3_CCR1T_X4:T_X7:T_X9:M_X1:M_X2:M_X8:M_X9 T_X2:M_X1:M_X6:M_X8 Inf CCL24_CCR2M_X6 T_X2:T_X6:M_X1:M_X2:M_X5:M_X6 Inf CCL22_DPP4 M_X7M_X4:M_X7:M_X10:M_X11 Inf CCL2_CCR5 M_X1:M_X6:M_X8 T_X2:M_X1:M_X6:M_X8Inf CCL2_CCR2 M_X1:M_X6:M_X8 T_X2:T_X6:M_X1:M_X2:M_X5:M_X6 Inf CCL2_CCR1M_X1:M_X6:M_X8 T_X2:M_X1:M_X6:M_X8 Inf CALM1_HMMR T_X4:T_X8:M_X7:M_X10T_X4:T_X8 Inf CADM1_CRTAM M_X10 T_X7:T_X9 Inf CADM1_CADM1 M_X10 M_X10Inf C5AR1_RPS19 M_X1:M_X6:M_X8:M_X9 T_X3:M_X0:M_X7:M_X12 Inf C5AR1_GNAI2M_X1:M_X6:M_X8:M_X9 T_X2:T_X8:M_X4:M_X11 Inf C3_IFITM1 M_X1:M_X5M_X4:M_X10:M_X11 Inf C3_CD81 M_X1:M_X5 T_X2:T_X8:M_X0:M_X12 Inf C3_CD19M_X1:M_X5 M_X0:M_X12 Inf C3_C3AR1 M_X1:M_X5 M_X1:M_X6:M_X8 Inf C1QB_LRP1M_X1:M_X6:M_X8 M_X1:M_X5:M_X6:M_X8:M_X9 Inf BTLA_CD79A M_X0:M_X10:M_X11M_X0:M_X12 Inf BTLA_CD247 M_X0:M_X10:M_X11 M_X2:M_X6 Inf B2M_TFRCT_X2:M_X6:M_X7:M_X9 T_X4:T_X9:M_X3 Inf B2M_KLRD1 T_X2:M_X6:M_X7:M_X9T_X0:T_X1:T_X4:T_X7:M_X2:M_X3:M_X4:M_X1 Inf 1 B2M_KLRC1T_X2:M_X6:M_X7:M_X9 T_X1:T_X4:T_X7:T_X9:M_X2 Inf B2M_HFET_X2:M_X6:M_X7:M_X9 M_X4:M_X5:M_X7:M_X9:M_X10 Inf B2M_CD3GT_X2:M_X6:M_X7:M_X9 T_X7:M_X6 Inf B2M_CD3D T_X2:M_X6:M_X7:M_X9 T_X7:M_X6Inf B2M_CD247 T_X2:M_X6:M_X7:M_X9 M_X2:M_X6 Inf APP_TNERSF21M_X1:M_X5:M_X6:M_X8 M_X1:M_X5 Inf APP_NGFR M_X1:M_X5:M_X6:M_X8 M_X11 InfAPP_LRP1 M_X1:M_X5:M_X6:M_X8 M_X1:M_X5:M_X6:M_X8:M_X9 Inf APP_CD74M_X1:M_X5:M_X6:M_X8 T_X2:T_X8:M_X0:M_X4:M_X7:M_X10 Inf APOE_SORL1M_X1:M_X5:M_X6:M_X8:M_X9 M_X9 Inf APOE_SCARB1 M_X1:M_X5:M_X6:M_X8:M_X9M_X5:M_X11 Inf APOE_LRP8 M_X1:M_X5:M_X6:M_X8:M_X9 M_X3 Inf APOE_LRP1M_X1:M_X5:M_X6:M_X8:M_X9 M_X1:M_X5:M_X6:M_X8:M_X9 Inf ALCAM_CD6T_X2:T_X9:M_X4:M_X11 T_X2:T_X6:M_X6 Inf ADRB2_IL1B T_X3:M_X0:M_X9M_X1:M_X4:M_X5:M_X6:M_X9 Inf FN1_ITGB8 M_X1:M_X5:M_X6:M_X8 T_X2 74254.31CALM3_SELL T_X4:T_X7:T_X8:M_X7 T_X0:T_X3:M_X2:M_X3:M_X11 67024.24IL18_1L18R1 M_X1 T_X6:M_X2 63600.53 FN1_PLAUR M_X1:M_X5:M_X6:M_X8M_X1:M_X3:M_X5:M_X8:M_X9 63175.69 PTPRC_CD22 M_X2:M_X9 M_X0 56364.05CD22_PTPRC M_X0 M_X2:M_X9 56364.05 FN1ITGB1 M_X1:M_X5:M_X6:M_X8T_X1:T_X5:T_X6:M_X2:M_X7 56109.13 CALM1_SELL T_X4:T_X8:M_X7:M_X10T_X0:T_X3:M_X2:M_X3:M_X11 53276.30 THBS1_SDC4 M_X1:M_X6:M_X8T_X2:M_X1:M_X3 52138.59 F10_ITGAM M_X1:M_X5:M_X6:M_X8:M_X9M_X1:M_X6:M_X8 51971.38 SEMA7A_ITGB1 M_X7 T_X1:T_X5:T_X6:M_X2:M_X751903.01 ITGB1_SEMA7A T_X1:T_X5:T_X6:M_X2:M_X7 M_X7 51903.01 CXCL10_SDC4M_X1:M_X4:M_X6 T_X2:M_X1:M_X3 50598.07 FN1_CD44 M_X1:M_X5:M_X6:M_X8T_X6:M_X1:M_X3:M_X5:M_X8:M_X9 50426.33 CD44_FN1T_X6:M_X1:M_X3:M_X5:M_X8:M_X9 M_X1:M_X5:M_X6:M_X8 50426.33 SPP1_ITGB1T_X4:T_X7:M_X1:M_X6:M_X8 T_X1:T_X5:T_X6:M_X2:M_X7 50066.54 ITGB1_SPP1T_X1:T_X5:T_X6:M_X2:M_X7 T_X4:T_X7:M_X1:M_X6:M_X8 50066.54 FN1_ITGAVM_X1:M_X5:M_X6:M_X8 T_X2 48656.13 THBS1_ITGB1 M_X1:M_X6:M_X8T_X1:T_X5:T_X6:M_X2:M_X7 48654.63 ITGB1_THBS1 T_X1:T_X5:T_X6:M_X2:M_X7M_X1:M_X6:M_X8 48654.63 CD40LG_ITGAM T_X5:T_X6 M_X1:M_X6:M_X8 47121.32TGM2_TBXA2R M_X1:M_X6:M_X8:M_X9 MX3 45503.40 SPP1_CD44T_X4:T_X7:M_X1:M_X6:M_X8 T_X6:M_X1:M_X3:M_X5:M_X8:M_X9 44995.74CD44_5PP1 T_X6:M_X1:M_X3:M_X5:M_X8:M_X9 T_X4:T_X7:M_X1:M_X6:M_X844995.74 F10_ITGB2 M_X1:M_X5:M_X6:M_X8:M_X9 T_X7:M_X1:M_X9 43769.00SPP1_ITGAV T_X4:T_X7:M_X1:M_X6:M_X8 T_X2 43416.19 ITGAV_SPP1 T_X2T_X4:T_X7:M_X1:M_X6:M_X8 43416.19 PTPRC_MRC1 M_X2:M_X9 M_X6:M_X843075.70 TGM2_SDC4 M_X1:M_X6:M_X8:M_X9 T_X2:M_X1:M_X3 41892.43 SDC4_TGM2T_X2:M_X1:M_X3 M_X1:M_X6:M_X8:M_X9 41892.43 LY86_CD180 M_X1:M_X3:M_X11M_X0:M_X3 41719.32 CD180_LY86 M_X0:M_X3 M_X1:M_X3:M_X11 41719.32IL18_1L18RAP M_X1 M_X2 40218.96 CD40LG_ITGB2 T_X5:T_X6 T_X7:M_X1:M_X939684.40 SEMA7A_PLXNC1 M_X7 M_X7 39644.16 PLXNC1_SEMA7A M_X7 M_X739644.16 TGM2_ITGB1 M_X1:M_X6:M_X8:M_X9 T_X1:T_X5:T_X6:M_X2:M_X739093.14 ITGB1_TGM2 T_X1:T_X5:T_X6:M_X2:M_X7 M_X1:M_X6:M_X8:M_X939093.14 CXCL2_DPP4 M_X1:M_X6:M_X8:M_X9 M_X4:M_X7:M_X10:M_X11 34788.81MMP12_PLAUR M_X8 M_X1:M_X3:M_X5:M_X8:M_X9 32846.29 CALR_ITGAVT_X2:T_X4:T_X7:T_X8:T_X9:M_X1:M_X4:M_X1 T_X2 0:M_X11 32261.62 SPP1_S1PR1T_X4:T_X7:M_X1:M_X6:M_X8 T_X3:M_X0 29430.92 S1PR1_SPP1 T_X3:M_X0T_X4:T_X7:M_X1:M_X6:M_X8 29430.92 TRF_GPR162 M_X1:M_X5 M_X3 29085.73CD70_CD27 T_X7:M_X7 T_X2:M_X2 28787.74 CD27_CD70 T_X2:M_X2 T_X7:M_X728787.74 LPL_CD44 M_X1:M_X6:M_X8 T_X6:M_X1:M_X3:M_X5:M_X8:M_X9 28622.36TGFB1_ITGB8 T_X2:T_X8:T_X9:M_X1:M_X5:M_X9 T_X2 28369.98 ITGB8_TGFB1 T_X2T_X2:T_X8:T_X9:M_X1:M_X5:M_X9 28369.98 C3_ITGAM M_X1:M_X5 M_X1:M_X6:M_X828337.51 THBS1_LRP1 M_X1:M_X6:M_X8 M_X1:M_X5:M_X6:M_X8:M_X9 26925.50LRP1_THBS1 M_X1:M_X5:M_X6:M_X8:M_X9 M_X1:MX6:M_X8 26925.50 LAMC1_ITGB1T_X2 T_X1:T_X5:T_X6:M_X2:M_X7 25224.82 CXCL10_CXCR3 M_X1:M_X4:M_X6T_X1:T_X2:T_X5:T_X6:M_X2:M_X3 24130.98 CALM2_SELL T_X8:M_X2T_X0:T_X3:M_X2:M_X3:M_X11 24052.87 C3_ITGB2 M_X1:M_X5 T_X7:M_X1:M_X923865.14 CDH1_CDH1 M_X3 M_X3 23700.60 CD274_CD80 M_X7:M_X8 M_X7 23029.97CCL8_CCR5 M_X8 T_X2:M_X1:M_X6:M_X8 22214.03 LAMC1_ITGAV T_X2 T_X221874.20 CD40LG_CD40 T_X5:T_X6 M_X1:M_X4:M_X7 21489.63 CD40_CD40LGM_X1:M_X4:M_X7 T_X5:T_X6 21489.63 THBS1_CD36 M_X1:M_X6:M_X8M_X6:M_X8:M_X9 21098.38 CD36_THBS1 M_X6:M_X8:M_X9 M_X1:M_X6:M_X821098.38 IL15_1L2RA M_X1:M_X7 T_X2:T_X8:T_X9:M_X7 20889.39 CCL1_CCR8T_X4:T_X7:T_X9 T_X2 20869.77 CALR_LRP1T_X2:T_X4:T_X7:T_X8:T_X9:M_X1:M_X4:M_X1 M_X1:M_X5:M_X6:M_X8:M_X920588.35 0:M_X11 IL18_CD48 M_X1 T_X1:T_X6:T_X7:M_X4:M_X11 18896.98TGFB1_ITGAV T_X2:T_X8:T_X9:M_X1:M_X5:M_X9 T_X2 18589.81 ITGAV_TGFB1 T_X2T_X2:T_X8:T_X9:M_X1:M_X5:M_X9 18589.81 C3_ITGAX M_X1:M_X5 M_X4:M_X1118384.81 CXCL9_DPP4 M_X1:M_X4:M_X6 M_X4:M_X7:M_X10:M_X11 18063.06ADAM9_ITGB1 M_X1 T_X1:T_X5:T_X6:M_X2:M_X7 17973.72 LPL_LRP1M_X1:M_X6:M_X8 M_X1:M_X5:M_X6:M_X8:M_X9 17624.68 CALM1_KCNN4T_X4:T_X8:M_X7:M_X10 T_X6:M_X4:M_X11 17430.67 C3_LRP1 M_X1:M_X5M_X1:M_X5:M_X6:M_X8:M_X9 17130.73 CALM1_FAS T_X4:T_X8:M_X7:M_X10M_X1:M_X7 17048.98 ADAM15_ITGB1 M_X1:M_X5 T_X1:T_X5:T_X6:M_X2:M_X716948.85 LGALS3BP_ITGB1 T_X6:M_X1:M_X6 T_X1:T_X5:T_X6:M_X2:M_X7 16694.11ITGB1_LGALS3BP T_X1:T_X5:T_X6:M_X2:M_X7 T_X6:M_X1:M_X6 16694.11ICAM1_IL2RA T_X2:T_X9:M_X1:M_X7 T_X2:T_X8:T_X9:M_X7 16398.94 ADAM9_ITGAVM_X1 T_X2 15586.26 THBS1_SCARB1 M_X1:M_X6:M_X8 M_X5:M_X11 15337.78THBS1_SDC1 M_X1:M_X6:M_X8 M_X8 15133.27 SDC1_THBS1 M_X8 M_X1:M_X6:M_X815133.27 FN1_NT5E M_X1:M_X5:M_X6:M_X8 T_X2 14948.61 FN1_ITGA4M_X1:M_X5:M_X6:M_X8 T_X1:M_X7:M_X9 14874.73 ADAM15_ITGAV M_X1:M_X5 T_X214697.53 GNAI2_TBXA2R T_X2:T_X8:M_X4:M_X11 M_X3 13917.42 LAMB3_ITGB1T_X1:M_X0:M_X2 T_X1:T_X5:T_X6:M_X2:M_X7 13360.82 SPP1_ITGA4T_X4:T_X7:M_X1:M_X6:M_X8 T_X1:M_X7:M_X9 13272.82 ITGA4_SPP1T_X1:M_X7:M_X9 T_X4:T_X7:M_X1:M_X6:M_X8 13272.82 GNAI2_CCR5T_X2:T_X8:M_X4:M_X11 T_X2:M_X1:M_X6:M_X8 13192.03 CCR5_GNAI2T_X2:M_X1:M_X6:M_X8 T_X2:T_X8:M_X4:M_X11 13192.03 THBS1_ITGA4M_X1:M_X6:M_X8 T_X1:M_X7:M_X9 12898.52 ITGA4_THBS1 T_X1:M_X7:M_X9M_X1:M_X6:M_X8 12898.52 IL15_1L15RA M_X1:M_X7 M_X7 11769.12 CXCL9_CXCR3M_X1:M_X4:M_X6 T_X1:T_X2:T_X5:T_X6:M_X2:M_X3 11586.63 ICAM1_ITGAMT_X2:T_X9:M_X1:M_X7 M_X1:M_X6:M_X8 10773.89 PF4_CXCR3 M_X6:M_X8T_X1:T_X2:T_X5:T_X6:M_X2:M_X3 10613.76 CXCR3_PF4T_X1:T_X2:T_X5:T_X6:M_X2:M_X3 M_X6:M_X8 10613.76 TGM2_ITGA4M_X1:M_X6:M_X8:M_X9 T_X1:M_X7:M_X9 10363.73 ANXA1_FPR2T_X6:M_X1:M_X5:M_X8:M_X10 M_X1 10005.94 LPL_SDC1 M_X1:M_X6:M_X8 M_X8 9905.82 PECAM1_PECAM T_X3:M_X0:M_X3 T_X3:M_X0:M_X3  9801.00 1 CFH_SELLM_X5 T_X0:T_X3:M_X2:M_X3:M_X11  9322.07 TGFB1_ACVRL1T_X2:T_X8:T_X9:M_X1:M_X5:M_X9 M_X4:M_X8:M_X10  9178.75 ACVRL1_TGFB1M_X4:M_X8:M_X10_ T_X2:T_X8:T_X9:M_X1:M_X5:M_X9  9178.75 CD48_CD244T_X1:T_X6:T_X7:M_X4:M_X11 T_X7:M_X2:M_X5  9169.75 VEGFA_ITGB1 M_X8T_X1:T_X5:T_X6:M_X2:M_X7  9081.65 ICAM1_ITGB2 T_X2:T_X9:M_X1:M_X7T_X7:M_X1:M_X9  9073.50 VEGFA_ITGAV M_X8 T_X2  7875.33 GPI1_AMFRT_X2:T_X7:M_X8 M_X3  7743.00 AMFR_GPI1 M_X3 T_X2:T_X7:M_X8  7743.00GNAI2_F2R T_X2:T_X8:M_X4:M_X11 M_X2  7562.70 F2R_GNAI2 M_X2T_X2:T_X8:M_X4:M_X11  7562.70 C1QA_CD93 M_X1:M_X6:M_X8 M_X8  7470.50GNAI2_S1PR1 T_X2:T_X8:M_X4:M_X11 T_X3:M_X0  7028.64 ICAM1_ITGAXT_X2:T_X9:M_X1:M_X7 M_X4:M_X11  6989.89 PF4_THBD M_X6:M_X8 M_X5  6952.79TGFB1_TGFBR1 T_X2:T_X8:T_X9:M_X1:M_X5:M_X9 MX3:M_X9_  6914.19GNAI2_CXCR3 T_X2:T_X8:M_X4:M_X11 T_X1:T_X2:T_X5:T_X6:M_X2:M_X3  6110.71VEGFA_SIRPA M_X8 M_X1:M_X3:M_X5  5691.26 L1CAM_ITGAV M_X11 T_X2  5597.84ITGAV_L1CAM T_X2 M_X11  5597.84 CFH_ITGAM M_X5 M_X1:M_X6:M_X8  5589.41GNAI2_S1PR4 T_X2:T_X8:M_X4:M_X11 T_X6:M_X2:M_X3  5476.14 TNFSF14_LTBRT_X9 M_X1:M_X5  5213.50 LTBR_TNFSF14 M_X1.M_X5 T_X9  5213.50UBA52_TGFBR1 T_X3:M_X0 M_X3M_X9  4667.20 ICAM1_ITGAL T_X2:T_X9:M_X1:M_X7M_X5:M_X9  4639.26 VEGFA_NRP1 M_X8 T_X6:T_X7:M_X3:M_X4  4543.81NRP1_VEGFA T_X6:T_X7:M_X3:M_X4 M_X8  4543.81 TNFSF4_TRAF2 M_X7 M_X7 4466.16 VEGFA_NRP2 M_X8 M_X7  4392.05 NRP2_VEGFA M_X7 M_X8  4392.05GNAI2_S1PR5 T_X2:T_X8:M_X4:M_X11 M_X9  4111.35 TGFB1_ENGT_X2:T_X8:T_X9:M_X1:M_X5:M_X9 M_X11  3334.96 ENG_TGFB1 M_X11T_X2:T_X8:T_X9:M_X1:M_X5:M_X9  3334.96 IL16_CCR5 M_X0:M_X2T_X2:M_X1:M_X6:M_X8  3176.54 TNFSF9_TRAF2 M_X4:M_X7:M_X11 M_X7  2241.62ICAM2_ITGAM M_X0 M_X1:M_X6:M_X8  2107.78 IL15_1L2RG M_X1:M_X7T_X6:M_X0:M_X2  2089.32 ICAM2_ITGB2 M_X0 T_X7:M_X1:M_X9  1775.12P4HB_GPR162 M_X8 M_X3  1774.24 ICAM1_IL2RG T_X2:T_X9:M_X1:M_X7T_X6:M_X0:M_X2  1640.19 TGFB1_CXCR4 T_X2:T_X8:T_X9:M_X1:M_X5:M_X9 M_X9 1346.14 TNESF11_TNERSF T_X9 M_X7  1225.30 11A TNERSF11A_TNES M_X7 T_X9 1225.30 F11 VEGFA_KDR M_X8 M_X11  1103.76 KDR_VEGFA M_X11 M_X8  1103.76L1CAM_L1CAM M_X11 M_X11  1068.64 ICAM2_ITGAL M_X0 M_X5:M_X9  907.61ADAM9_ITGB5 M_X1 M_X9  242.11 SEMA4D_CD72 M_X9 M_X0:M_X3:M_X12   82.80CD72_SEMA4D M_X0:M_X3:M_X12 M_X9   82.80 SEMA4D_PLXNB2 M_X9 M_X9   1.01PLXNB2_SEMA4D M_X9 M_X9   1.01Identification of Cell Types and Genes Correlated with Tumor Size andTime

In certain embodiments, cell types in the TME and genes expressed in thecell types change during tumor progression, such as with time and tumorsize. In certain embodiments, cell types capable of signaling other celltypes in the TME are positively or negatively correlated with time andsize. In certain embodiments, signaling molecules expressed in specificcell types are positively or negatively correlated with time and size.In certain embodiments, signaling from the cell types suppress orenhance anti-tumor immunity. In certain embodiments, the cells types orgenes can be detected or modified.

Signature Genes

In certain embodiments, cell types of interacting cells are identifiedby gene signatures. In certain embodiments, genes associated with tumorprogression (e.g., time or size) are part of a gene signature. Incertain embodiments, genes in a gene signature are associated withsimilar pathways. As used herein a “signature” may encompass any gene orgenes, protein or proteins, or epigenetic element(s) whose expressionprofile or whose occurrence is associated with a specific cell type,subtype, or cell state of a specific cell type or subtype within apopulation of cells. For ease of discussion, when discussing geneexpression, any of gene or genes, protein or proteins, or epigeneticelement(s) may be substituted. As used herein, the terms “signature”,“expression profile”, or “expression program” may be usedinterchangeably. As used herein the term “biological program” or “cellprogram” may be a type of “signature”, “expression program” or“transcriptional program” and refers to a set of genes that share a rolein a biological function (e.g., an activation program, celldifferentiation program, proliferation program). Biological programs caninclude a pattern of gene expression that result in a correspondingphysiological event or phenotypic trait. Biological programs can includeup to several hundred genes that are expressed in a spatially andtemporally controlled fashion. Expression of individual genes can beshared between biological programs. Expression of individual genes canbe shared among different single cell types; however, expression of abiological program may be cell type specific or temporally specific(e.g., the biological program is expressed in a cell type at a specifictime). Biological programs may be expressed across different cell types.In certain embodiments, a biological program includes genes thatco-vary. Expression of a biological program may be regulated by a masterswitch, such as a nuclear receptor or transcription factor. As usedherein, the term “topic” refers to a biological program. The biologicalprogram (e.g., topics) can be modeled as a distribution over expressedgenes. One method to identify cell programs is non-negative matrixfactorization (NMF) (see, e.g., Lee D D and Seung H S, Learning theparts of objects by non-negative matrix factorization, Nature. 1999 Oct.21; 401(6755):788-91). Other approaches are topic models (Bielecki,Riesenfeld, Kowalczyk, et al., 2018 Skin inflammation driven bydifferentiation of quiescent tissue-resident ILCs into a spectrum ofpathogenic effectors. bioRxiv 461228) and word embeddings. Identifyingcell programs can recover cell states and bridge differences betweencells. Single cell types may span a range of continuous cell states(see, e.g., Shekhar et al., Comprehensive Classification of RetinalBipolar Neurons by Single-Cell Transcriptomics Cell. 2016 Aug. 25;166(5):1308-1323.e30; and Bielecki, et al., 2018 bioRxiv 461228).

It is to be understood that also when referring to proteins (e.g.differentially expressed proteins), such may fall within the definitionof “gene” signature. Levels of expression or activity or prevalence maybe compared between different cells in order to characterize or identifyfor instance signatures specific for cell (sub)populations. Increased ordecreased expression or activity or prevalence of signature genes may becompared between different cells in order to characterize or identifyfor instance specific cell (sub)populations. The detection of asignature in single cells may be used to identify and quantitate forinstance specific cell (sub)populations. A signature may include a geneor genes, protein or proteins, or epigenetic element(s) whose expressionor occurrence is specific to a cell (sub)population, such thatexpression or occurrence is exclusive to the cell (sub)population. Agene signature as used herein, may thus refer to any set of up- anddown-regulated genes that are representative of a cell type or subtype.A gene signature as used herein, may also refer to any set of up- anddown-regulated genes between different cells or cell (sub)populationsderived from a gene-expression profile. For example, a gene signaturemay comprise a list of genes differentially expressed in a distinctionof interest.

The signature as defined herein (being it a gene signature, proteinsignature or other genetic or epigenetic signature) can be used toindicate the presence of a cell type, a subtype of the cell type, thestate of the microenvironment of a population of cells, a particularcell type population or subpopulation, and/or the overall status of theentire cell (sub)population. Furthermore, the signature may beindicative of cells within a population of cells in vivo. The signaturemay also be used to suggest for instance particular therapies, or tofollow up treatment, or to suggest ways to modulate immune systems. Thesignatures of the present invention may be discovered by analysis ofexpression profiles of single-cells within a population of cells fromisolated samples (e.g. tumor samples), thus allowing the discovery ofnovel cell subtypes or cell states that were previously invisible orunrecognized. The presence of subtypes or cell states may be determinedby subtype specific or cell state specific signatures. The presence ofthese specific cell (sub)types or cell states may be determined byapplying the signature genes to bulk sequencing data in a sample. Notbeing bound by a theory the signatures of the present invention may bemicroenvironment specific, such as their expression in a particularspatio-temporal context. Not being bound by a theory, signatures asdiscussed herein are specific to a particular pathological context. Notbeing bound by a theory, a combination of cell subtypes having aparticular signature may indicate an outcome. Not being bound by atheory, the signatures can be used to deconvolute the network of cellspresent in a particular pathological condition. Not being bound by atheory the presence of specific cells and cell subtypes are indicativeof a particular response to treatment, such as including increased ordecreased susceptibility to treatment. The signature may indicate thepresence of one particular cell type. In one embodiment, the novelsignatures are used to detect multiple cell states or hierarchies thatoccur in subpopulations of cancer cells that are linked to particularpathological condition (e.g. cancer grade), or linked to a particularoutcome or progression of the disease (e.g. metastasis), or linked to aparticular response to treatment of the disease.

The signature according to certain embodiments of the present inventionmay comprise or consist of one or more genes, proteins and/or epigeneticelements, such as for instance 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Incertain embodiments, the signature may comprise or consist of two ormore genes, proteins and/or epigenetic elements, such as for instance 2,3, 4, 5, 6, 7, 8, 9, 10 or more. In certain embodiments, the signaturemay comprise or consist of three or more genes, proteins and/orepigenetic elements, such as for instance 3, 4, 5, 6, 7, 8, 9, 10 ormore. In certain embodiments, the signature may comprise or consist offour or more genes, proteins and/or epigenetic elements, such as forinstance 4, 5, 6, 7, 8, 9, 10 or more. In certain embodiments, thesignature may comprise or consist of five or more genes, proteins and/orepigenetic elements, such as for instance 5, 6, 7, 8, 9, 10 or more. Incertain embodiments, the signature may comprise or consist of six ormore genes, proteins and/or epigenetic elements, such as for instance 6,7, 8, 9, 10 or more. In certain embodiments, the signature may compriseor consist of seven or more genes, proteins and/or epigenetic elements,such as for instance 7, 8, 9, 10 or more. In certain embodiments, thesignature may comprise or consist of eight or more genes, proteinsand/or epigenetic elements, such as for instance 8, 9, 10 or more. Incertain embodiments, the signature may comprise or consist of nine ormore genes, proteins and/or epigenetic elements, such as for instance 9,10 or more. In certain embodiments, the signature may comprise orconsist of ten or more genes, proteins and/or epigenetic elements, suchas for instance 10, 11, 12, 13, 14, 15, or more. It is to be understoodthat a signature according to the invention may for instance alsoinclude genes or proteins as well as epigenetic elements combined.

In certain embodiments, a signature is characterized as being specificfor a particular cell or cell (sub)population if it is upregulated oronly present, detected or detectable in that particular tumor cell ortumor cell (sub)population, or alternatively is downregulated or onlyabsent, or undetectable in that particular tumor cell or tumor cell(sub)population. In this context, a signature consists of one or moredifferentially expressed genes/proteins or differential epigeneticelements when comparing different cells or cell (sub)populations,including comparing different cells or cell (sub)populations, as well ascomparing tumor cells or tumor cell (sub)populations with non-tumorcells or non-tumor cell (sub)populations. It is to be understood that“differentially expressed” genes/proteins include genes/proteins whichare up- or down-regulated as well as genes/proteins which are turned onor off. When referring to up-or down-regulation, in certain embodiments,such up- or down-regulation is preferably at least two-fold, such astwo-fold, three-fold, four-fold, five-fold, or more, such as forinstance at least ten-fold, at least 20-fold, at least 30-fold, at least40-fold, at least 50-fold, or more. Alternatively, or in addition,differential expression may be determined based on common statisticaltests, as is known in the art.

As discussed herein, differentially expressed genes/proteins, ordifferential epigenetic elements may be differentially expressed on asingle cell level, or may be differentially expressed on a cellpopulation level. Preferably, the differentially expressed genes/proteins or epigenetic elements as discussed herein, such asconstituting the gene signatures as discussed herein, when as to thecell population level, refer to genes that are differentially expressedin all or substantially all cells of the population (such as at least80%, preferably at least 90%, such as at least 95% of the individualcells). This allows one to define a particular subpopulation of tumorcells. As referred to herein, a “subpopulation” of cells preferablyrefers to a particular subset of cells of a particular cell type whichcan be distinguished or are uniquely identifiable and set apart fromother cells of this cell type. The cell subpopulation may bephenotypically characterized, and is preferably characterized by thesignature as discussed herein. A cell (sub)population as referred toherein may constitute of a (sub)population of cells of a particular celltype characterized by a specific cell state.

When referring to induction, or alternatively suppression of aparticular signature, preferable is meant induction or alternativelysuppression (or upregulation or downregulation) of at least onegene/protein and/or epigenetic element of the signature, such as forinstance at least to, at least three, at least four, at least five, atleast six, or all genes/proteins and/or epigenetic elements of thesignature.

Signatures may be functionally validated as being uniquely associatedwith a particular immune responder phenotype. Induction or suppressionof a particular signature may consequentially be associated with orcausally drive a particular immune responder phenotype.

Various aspects and embodiments of the invention may involve analyzinggene signatures, protein signature, and/or other genetic or epigeneticsignature based on single cell analyses (e.g. single cell RNAsequencing) or alternatively based on cell population analyses, as isdefined herein elsewhere.

In further aspects, the invention relates to gene signatures, proteinsignature, and/or other genetic or epigenetic signature of particulartumor cell subpopulations, as defined herein elsewhere. The inventionhereto also further relates to particular tumor cell subpopulations,which may be identified based on the methods according to the inventionas discussed herein; as well as methods to obtain such cell(sub)populations and screening methods to identify agents capable ofinducing or suppressing particular tumor cell (sub)populations.

The invention further relates to various uses of the gene signatures,protein signature, and/or other genetic or epigenetic signature asdefined herein, as well as various uses of the tumor cells or tumor cell(sub)populations as defined herein. Particular advantageous uses includemethods for identifying agents capable of inducing or suppressingparticular tumor cell (sub)populations based on the gene signatures,protein signature, and/or other genetic or epigenetic signature asdefined herein. The invention further relates to agents capable ofinducing or suppressing particular tumor cell (sub)populations based onthe gene signatures, protein signature, and/or other genetic orepigenetic signature as defined herein, as well as their use formodulating, such as inducing or repressing, a particular gene signature,protein signature, and/or other genetic or epigenetic signature. In oneembodiment, genes in one population of cells may be activated orsuppressed in order to affect the cells of another population. Inrelated aspects, modulating, such as inducing or repressing, aparticular a particular gene signature, protein signature, and/or othergenetic or epigenetic signature may modify overall tumor composition,such as tumor cell composition, such as tumor cell subpopulationcomposition or distribution, or functionality.

The signature genes of the present invention were discovered by analysisof expression profiles of single-cells within a population of cells,thus allowing the discovery of novel cell subtypes that were previouslyinvisible in a population of cells within a tissue. The presence ofsubtypes may be determined by subtype specific signature genes. Thepresence of these specific cell types may be determined by applying thesignature genes to bulk sequencing data in a patient tumor. Not beingbound by a theory, a tumor is a conglomeration of many cells that makeup a tumor microenvironment, whereby the cells communicate and affecteach other in specific ways. As such, specific cell types within thismicroenvironment may express signature genes specific for thismicroenvironment. Not being bound by a theory the signature genes of thepresent invention may be microenvironment specific, such as theirexpression in a tumor. Not being bound by a theory, signature genesdetermined in single cells that originated in a tumor are specific toother tumors. Not being bound by a theory, a combination of cellsubtypes in a tumor may indicate an outcome. Not being bound by atheory, the signature genes can be used to deconvolute the network ofcells present in a tumor based on comparing them to data from bulkanalysis of a tumor sample. Not being bound by a theory the presence ofspecific cells and cell subtypes may be indicative of tumor growth,invasiveness and resistance to treatment. The signature gene mayindicate the presence of one particular cell type. In one embodiment,the signature genes may indicate that tumor infiltrating T-cells arepresent. The presence of cell types within a tumor may indicate that thetumor will be resistant to a treatment. In one embodiment, the signaturegenes of the present invention are applied to bulk sequencing data froma tumor sample obtained from a subject, such that information relatingto disease outcome and personalized treatments is determined. In oneembodiment, the novel signature genes are used to detect multiple cellstates that occur in a subpopulation of tumor cells that are linked toresistance to targeted therapies and progressive tumor growth.

All gene name symbols refer to the gene as commonly known in the art.The examples described herein that refer to the mouse gene names are tobe understood to also encompasses human genes, as well as genes in anyother organism (e.g., homologous, orthologous genes). Mouse gene symbolsare generally italicized, with only the first letter in upper-case(e.g., Nrp1). Mouse protein symbols are generally not italicized, andall letters are in upper-case (e.g., NRP1). As used herein mouse genesymbols may be shown with only the first letter in upper-case and notitalicized (e.g., Nrp1). Any reference to the gene symbol is a referencemade to the entire gene or variants of the gene. Any reference to thegene symbol is also a reference made to the gene product (e.g.,protein). The term, homolog, may apply to the relationship between genesseparated by the event of speciation (e.g., ortholog). Orthologs aregenes in different species that evolved from a common ancestral gene byspeciation. Normally, orthologs retain the same function in the courseof evolution. Gene symbols may be those referred to by the HUGO GeneNomenclature Committee (HGNC) or National Center for BiotechnologyInformation (NCBI). The signature as described herein may encompass anyof the genes described herein.

Interacting proteins, such as ligand receptor pairs, may be searched forin public databases (see, e.g., Giurgiu et al., CORUM: the comprehensiveresource of mammalian protein complexes-2019. Nucleic Acids Res. 2019Jan. 8; 47(D1):D559-D563; Ramilowski, J. A., Goldberg, T., Harshbarger,J., Kloppmann, E., Lizio, M., Satagopam, V. P., Itoh, M., Kawaji, H.,Carninci, P., Rost, B., et al. (2015). A draft network ofligand-receptor-mediated multicellular signalling in human. Nat Commun6, 7866; and dip.doe-mbi.ucla.edu/dip/DLRP.cgi).

Applicants have identified interactions between tumor cells and immunecells, and between immune cells. The interactions can modulate immuneresponses in the tumor microenvironment. Tumors can interact with immunecells to suppress an immune response. Tumor cells may activatesuppressive immune cells or suppress effector immune cells.

Modified CD8 T-Cells and Therapeutic Uses Thereof

In certain example embodiments, the ligand-receptor pairs disclosedherein are used to generated modified T cells for therapeutic use, forexample in adoptive cell therapeutic use. The modifications disclosedherein generally enhance T cell function by removing a ligand orreceptor that allows for communication with another immune cell thatwould signal the T cell to have a suppressed immune response. In certainexample embodiments, CD8 T cells are modified to decrease or eliminateexpression, activity or function of NPR1 to prevent communicationbetween the tumor cells and the T cell. In another example embodiment,CD8 T cells are modified to decrease or eliminate expression activity,or function of CRTAM. In certain embodiments, the modified T cells arefurther modified to express a T cell receptor or chimeric antigenreceptor specific for a tumor specific antigen. In certain embodiments,the modified T cells are transferred to a subject in need thereof totreat a tumor.

Adoptive Cell Transfer

In certain embodiments, the methods of the present invention may be usedto modulate interacting immune cells ex vivo for transfer into asubject. In certain embodiments, modulating one or more identifiedtherapeutic targets in an immune cell shifts the immune cell to beresistant to dysfunction or have increased effector function (e.g.,NRP1, CRTAM). Such immune cells may be used to increase theeffectiveness of adoptive cell transfer. In certain embodiments, immunecells are modulated using a genetic modifying agent, antibody or smallmolecule, described further herein.

As used herein, “ACT”, “adoptive cell therapy” and “adoptive celltransfer” may be used interchangeably. In certain embodiments, Adoptivecell therapy (ACT) can refer to the transfer of cells to a patient withthe goal of transferring the functionality and characteristics into thenew host by engraftment of the cells (see, e.g., Mettananda et al.,Editing an a-globin enhancer in primary human hematopoietic stem cellsas a treatment for β-thalassemia, Nat Commun. 2017 Sep. 4; 8(1):424). Asused herein, the term “engraft” or “engraftment” refers to the processof cell incorporation into a tissue of interest in vivo through contactwith existing cells of the tissue. Adoptive cell therapy (ACT) can referto the transfer of cells, most commonly immune-derived cells, back intothe same patient or into a new recipient host with the goal oftransferring the immunologic functionality and characteristics into thenew host. If possible, use of autologous cells helps the recipient byminimizing GVHD issues. The adoptive transfer of autologous tumorinfiltrating lymphocytes (TIL) (Zacharakis et al., (2018) Nat Med. 2018June; 24(6):724-730; Besser et al., (2010) Clin. Cancer Res 16 (9)2646-55; Dudley et al., (2002) Science 298 (5594): 850-4; and Dudley etal., (2005) Journal of Clinical Oncology 23 (10): 2346-57.) orgenetically re-directed peripheral blood mononuclear cells (Johnson etal., (2009) Blood 114 (3): 535-46; and Morgan et al., (2006) Science314(5796) 126-9) has been used to successfully treat patients withadvanced solid tumors, including melanoma, metastatic breast cancer andcolorectal carcinoma, as well as patients with CD19-expressinghematologic malignancies (Kalos et al., (2011) Science TranslationalMedicine 3 (95): 95ra73). In certain embodiments, allogenic cells immunecells are transferred (see, e.g., Ren et al., (2017) Clin Cancer Res 23(9) 2255-2266). As described further herein, allogenic cells can beedited to reduce alloreactivity and prevent graft-versus-host disease.Thus, use of allogenic cells allows for cells to be obtained fromhealthy donors and prepared for use in patients as opposed to preparingautologous cells from a patient after diagnosis.

Aspects of the invention involve the adoptive transfer of immune systemcells, such as T cells, specific for selected antigens, such as tumorassociated antigens or tumor specific neoantigens (see, e.g., Maus etal., 2014, Adoptive Immunotherapy for Cancer or Viruses, Annual Reviewof Immunology, Vol. 32: 189-225; Rosenberg and Restifo, 2015, Adoptivecell transfer as personalized immunotherapy for human cancer, ScienceVol. 348 no. 6230 pp. 62-68; Restifo et al., 2015, Adoptiveimmunotherapy for cancer: harnessing the T cell response. Nat. Rev.Immunol. 12(4): 269-281; and Jenson and Riddell, 2014, Design andimplementation of adoptive therapy with chimeric antigenreceptor-modified T cells. Immunol Rev. 257(1): 127-144; and Rajasagi etal., 2014, Systematic identification of personal tumor-specificneoantigens in chronic lymphocytic leukemia. Blood. 2014 Jul. 17;124(3):453-62).

In certain embodiments, an antigen (such as a tumor antigen) to betargeted in adoptive cell therapy (such as particularly CAR or TCRT-cell therapy) of a disease (such as particularly of tumor or cancer)may be selected from a group consisting of: B cell maturation antigen(BCMA) (see, e.g., Friedman et al., Effective Targeting of MultipleBCMA-Expressing Hematological Malignancies by Anti-BCMA CAR T Cells, HumGene Ther. 2018 Mar 8; Berdeja J G, et al. Durable clinical responses inheavily pretreated patients with relapsed/refractory multiple myeloma:updated results from a multicenter study of bb2121 anti-Bcma CAR T celltherapy. Blood. 2017;130:740; and Mouhieddine and Ghobrial,Immunotherapy in Multiple Myeloma: The Era of CAR T Cell Therapy,Hematologist, May-June 2018, Volume 15, issue 3); PSA (prostate-specificantigen); prostate-specific membrane antigen (PSMA); PSCA (Prostate stemcell antigen); Tyrosine-protein kinase transmembrane receptor ROR1;fibroblast activation protein (FAP); Tumor-associated glycoprotein 72(TAG72); Carcinoembryonic antigen (CEA); Epithelial cell adhesionmolecule (EPCAM); Mesothelin; Human Epidermal growth factor Receptor 2(ERBB2 (Her2/neu)); Prostate; Prostatic acid phosphatase (PAP);elongation factor 2 mutant (ELF2M); Insulin-like growth factor 1receptor (IGF-1R); gplOO; BCR-ABL (breakpoint cluster region-Abelson);tyrosinase; New York esophageal squamous cell carcinoma 1 (NY-ESO-1);K-light chain, LAGE (L antigen); MAGE (melanoma antigen);Melanoma-associated antigen 1 (MAGE-A1); MAGE A3; MAGE A6; legumain;Human papillomavirus (HPV) E6; HPV E7; prostein; survivin; PCTA1(Galectin 8); Melan-A/MART-1; Ras mutant; TRP-1 (tyrosinase relatedprotein 1, or gp75); Tyrosinase-related Protein 2 (TRP2); TRP-2/INT2(TRP-2/intron 2); RAGE (renal antigen); receptor for advanced glycationend products 1 (RAGE1); Renal ubiquitous 1, 2 (RU1, RU2); intestinalcarboxyl esterase (iCE); Heat shock protein 70-2 (HSP70-2) mutant;thyroid stimulating hormone receptor (TSHR); CD123; CD171; CD19; CD20;CD22; CD26; CD30; CD33; CD44v7/8 (cluster of differentiation 44, exons7/8); CD53; CD92; CD100; CD148; CD150; CD200; CD261; CD262; CD362; CS-1(CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-likemolecule-1 (CLL-1); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDG1cp(1-1)Cer); Tn antigen (Tn Ag);Fms-Like Tyrosine Kinase 3 (FLT3); CD38; CD138; CD44v6; B7H3 (CD276);KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2);Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen(PSCA); Protease Serine 21 (PRSS21); vascular endothelial growth factorreceptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growthfactor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4(SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16 (MUC16);epidermal growth factor receptor (EGFR); epidermal growth factorreceptor variant III (EGFRvIII); neural cell adhesion molecule (NCAM);carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit,Beta Type, 9 (LMP2); ephrin type-A receptor 2 (EphA2); Ephrin B2;Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3(aNeu5Ac(2-3)bDGalp(1-4)bDG1cp(1-1)Cer); TGS5; high molecularweight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside(OAcGD2); Folate receptor alpha; Folate receptor beta; tumor endothelialmarker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R);claudin 6 (CLDN6); G protein-coupled receptor class C group 5, member D(GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a;anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1(PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH);mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2);Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3(ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2(OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumorprotein (WT1); ETS translocation-variant gene 6, located on chromosome12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A(XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); CT(cancer/testis (antigen)); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53;p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin B 1; Cyclin D1; v-myc avian myelocytomatosisviral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog FamilyMember C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor(Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma AntigenRecognized By T Cells-1 or 3 (SART1, SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (0Y-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint-1,-2,-3 or -4 (SSX1, SSX2, SSX3, SSX4);CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1(LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300molecule-like family member f (CD300LF); C-type lectin domain family 12member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyteantigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); mousedouble minute 2 homolog (MDM2); livin; alphafetoprotein (AFP);transmembrane activator and CAML Interactor (TACI); B-cell activatingfactor receptor (BAFF-R); V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog (KRAS); immunoglobulin lambda-like polypeptide 1 (IGLL1); 707-AP(707 alanine proline); ART-4 (adenocarcinoma antigen recognized by T4cells); BAGE (B antigen; b-catenin/m, b-catenin/mutated); CAMEL(CTL-recognized antigen on melanoma); CAP1 (carcinoembryonic antigenpeptide 1); CASP-8 (caspase-8); CDC27m (cell-division cycle 27 mutated);CDK4/m (cycline-dependent kinase 4 mutated); Cyp-B (cyclophilin B); DAM(differentiation antigen melanoma); EGP-2 (epithelial glycoprotein 2);EGP-40 (epithelial glycoprotein 40); Erbb2, 3, 4 (erythroblasticleukemia viral oncogene homolog-2,-3, 4); FBP (folate binding protein);, fAchR (Fetal acetylcholine receptor); G250 (glycoprotein 250); GAGE (Gantigen); GnT-V (N-acetylglucosaminyltransferase V); HAGE (helicoseantigen); ULA-A (human leukocyte antigen-A); HST2 (human signet ringtumor 2); KIAA0205; KDR (kinase insert domain receptor); LDLR/FUT (lowdensity lipid receptor/GDP L-fucose: b-D-galactosidase 2-a-Lfucosyltransferase); L1CAM (L1 cell adhesion molecule); MC1R(melanocortin 1 receptor); Myosin/m (myosin mutated); MUM-1,-2,-3(melanoma ubiquitous mutated 1, 2, 3); NA88-A (NA cDNA clone of patientM88); KG2D (Natural killer group 2, member D) ligands; oncofetal antigen(h5T4); p190 minor bcr-abl (protein of 190KD bcr-abl); Pml/RARa(promyelocytic leukaemia/retinoic acid receptor a); PRAME(preferentially expressed antigen of melanoma); SAGE (sarcoma antigen);TEL/AML1 (translocation Ets-family leukemia/acute myeloid leukemia 1);TPI/m (triosephosphate isomerase mutated); CD70; and any combinationthereof.

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a tumor-specific antigen(TSA).

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a neoantigen.

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a tumor-associated antigen(TAA).

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a universal tumor antigen.In certain preferred embodiments, the universal tumor antigen isselected from the group consisting of: a human telomerase reversetranscriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2),cytochrome P450 1B 1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1),livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16(MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin(Dl), and any combinations thereof.

In certain embodiments, an antigen (such as a tumor antigen) to betargeted in adoptive cell therapy (such as particularly CAR or TCRT-cell therapy) of a disease (such as particularly of tumor or cancer)may be selected from a group consisting of: CD19, BCMA, CD70, CLL-1,MAGE A3, MAGE A6, HPV E6, HPV E7, WT1, CD22, CD171, ROR1, MUC16, andSSX2. In certain preferred embodiments, the antigen may be CD19. Forexample, CD19 may be targeted in hematologic malignancies, such as inlymphomas, more particularly in B-cell lymphomas, such as withoutlimitation in diffuse large B-cell lymphoma, primary mediastinal b-celllymphoma, transformed follicular lymphoma, marginal zone lymphoma,mantle cell lymphoma, acute lymphoblastic leukemia including adult andpediatric ALL, non-Hodgkin lymphoma, indolent non-Hodgkin lymphoma, orchronic lymphocytic leukemia. For example, BCMA may be targeted inmultiple myeloma or plasma cell leukemia (see, e.g., 2018 AmericanAssociation for Cancer Research (AACR) Annual meeting Poster: AllogeneicChimeric Antigen Receptor T Cells Targeting B Cell Maturation Antigen).For example, CLL1 may be targeted in acute myeloid leukemia. Forexample, MAGE A3, MAGE A6, SSX2, and/or KRAS may be targeted in solidtumors. For example, HPV E6 and/or HPV E7 may be targeted in cervicalcancer or head and neck cancer. For example, WT1 may be targeted inacute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronicmyeloid leukemia (CIVIL), non-small cell lung cancer, breast,pancreatic, ovarian or colorectal cancers, or mesothelioma. For example,CD22 may be targeted in B cell malignancies, including non-Hodgkinlymphoma, diffuse large B-cell lymphoma, or acute lymphoblasticleukemia. For example, CD171 may be targeted in neuroblastoma,glioblastoma, or lung, pancreatic, or ovarian cancers. For example, ROR1may be targeted in ROR1+ malignancies, including non-small cell lungcancer, triple negative breast cancer, pancreatic cancer, prostatecancer, ALL, chronic lymphocytic leukemia, or mantle cell lymphoma. Forexample, MUC16 may be targeted in MUC16ecto+ epithelial ovarian,fallopian tube or primary peritoneal cancer. For example, CD70 may betargeted in both hematologic malignancies as well as in solid cancerssuch as renal cell carcinoma (RCC), gliomas (e.g., GBM), and head andneck cancers (HNSCC). CD70 is expressed in both hematologic malignanciesas well as in solid cancers, while its expression in normal tissues isrestricted to a subset of lymphoid cell types (see, e.g., 2018 AmericanAssociation for Cancer Research (AACR) Annual meeting Poster: AllogeneicCRISPR Engineered Anti-CD70 CAR-T Cells Demonstrate Potent PreclinicalActivity Against Both Solid and Hematological Cancer Cells).

Various strategies may for example be employed to genetically modify Tcells by altering the specificity of the T cell receptor (TCR) forexample by introducing new TCR α and β chains with selected peptidespecificity (see U.S. Pat. No. 8,697,854; PCT Patent Publications:WO2003020763, WO2004033685, WO2004044004, WO2005114215, WO2006000830,WO2008038002, WO2008039818, WO2004074322, WO2005113595, WO2006125962,WO2013166321, WO2013039889, WO2014018863, WO2014083173; U.S. Pat. No.8,088,379).

As an alternative to, or addition to, TCR modifications, chimericantigen receptors (CARs) may be used in order to generateimmunoresponsive cells, such as T cells, specific for selected targets,such as malignant cells, with a wide variety of receptor chimeraconstructs having been described (see U.S. Pat. Nos. 5,843,728;5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014;6,753,162; 8,211,422; and, PCT Patent Publication WO9215322).

In general, CARs are comprised of an extracellular domain, atransmembrane domain, and an intracellular domain, wherein theextracellular domain comprises an antigen-binding domain that isspecific for a predetermined target. While the antigen-binding domain ofa CAR is often an antibody or antibody fragment (e.g., a single chainvariable fragment, scFv), the binding domain is not particularly limitedso long as it results in specific recognition of a target. For example,in some embodiments, the antigen-binding domain may comprise a receptor,such that the CAR is capable of binding to the ligand of the receptor.Alternatively, the antigen-binding domain may comprise a ligand, suchthat the CAR is capable of binding the endogenous receptor of thatligand.

The antigen-binding domain of a CAR is generally separated from thetransmembrane domain by a hinge or spacer. The spacer is also notparticularly limited, and it is designed to provide the CAR withflexibility. For example, a spacer domain may comprise a portion of ahuman Fc domain, including a portion of the CH3 domain, or the hingeregion of any immunoglobulin, such as IgA, IgD, IgE, IgG, or IgM, orvariants thereof. Furthermore, the hinge region may be modified so as toprevent off-target binding by FcRs or other potential interferingobjects. For example, the hinge may comprise an IgG4 Fc domain with orwithout a S228P, L235E, and/or N297Q mutation (according to Kabatnumbering) in order to decrease binding to FcRs. Additionalspacers/hinges include, but are not limited to, CD4, CD8, and CD28 hingeregions.

The transmembrane domain of a CAR may be derived either from a naturalor from a synthetic source. Where the source is natural, the domain maybe derived from any membrane bound or transmembrane protein.Transmembrane regions of particular use in this disclosure may bederived from CD8, CD28, CD3, CD45, CD4, CD5, CDS, CD9, CD 16, CD22,CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, TCR. Alternatively,the transmembrane domain may be synthetic, in which case it willcomprise predominantly hydrophobic residues such as leucine and valine.Preferably a triplet of phenylalanine, tryptophan and valine will befound at each end of a synthetic transmembrane domain. Optionally, ashort oligo- or polypeptide linker, preferably between 2 and 10 aminoacids in length may form the linkage between the transmembrane domainand the cytoplasmic signaling domain of the CAR. A glycine-serinedoublet provides a particularly suitable linker.

Alternative CAR constructs may be characterized as belonging tosuccessive generations. First-generation CARs typically consist of asingle-chain variable fragment of an antibody specific for an antigen,for example comprising a VL linked to a VH of a specific antibody,linked by a flexible linker, for example by a CD8a hinge domain and aCD8a transmembrane domain, to the transmembrane and intracellularsignaling domains of either CD3 or FcRγ (scFv-CD3t or scFv-FcRγ; seeU.S. Pat. Nos. 7,741,465; 5,912,172; 5,906,936). Second-generation CARsincorporate the intracellular domains of one or more costimulatorymolecules, such as CD28, 0X40 (CD134), or 4-1BB (CD137) within theendodomain (for example scFv-CD28/OX40/4-1BB-CD3; see U.S. Pat. Nos.8,911,993; 8,916,381; 8,975,071; 9,101,584; 9,102,760; 9,102,761).Third-generation CARs include a combination of costimulatoryendodomains, such a CD3ζ-chain, CD97, GDI 1a-CD18, CD2, ICOS, CD27,CD154, CDS, OX40, 4-1BB, CD2, CD7, LIGHT, LFA-1, NKG2C, B7-H3, CD30,CD40, PD-1, or CD28 signaling domains (for example scFv-CD28-4-1BB-CD3ζor scFv-CD28-OX40-CD3ζ; see U.S. Pat. Nos. 8,906,682; 8,399,645;5,686,281; PCT Publication No. WO2014134165; PCT Publication No.WO2012079000). In certain embodiments, the primary signaling domaincomprises a functional signaling domain of a protein selected from thegroup consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, commonFcR gamma (FCERIG), FcR beta (Fc Epsilon R1b), CD79a, CD79b, Fc gammaRIM, DAP10, and DAP12. In certain preferred embodiments, the primarysignaling domain comprises a functional signaling domain of CD3ζ orFcRγ. In certain embodiments, the one or more costimulatory signalingdomains comprise a functional signaling domain of a protein selected,each independently, from the group consisting of: CD27, CD28, 4-1BB(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand thatspecifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR),SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta,IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD1 1 a, LFA-1, ITGAM,CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46,and NKG2D. In certain embodiments, the one or more costimulatorysignaling domains comprise a functional signaling domain of a proteinselected, each independently, from the group consisting of: 4-1BB, CD27,and CD28. In certain embodiments, a chimeric antigen receptor may havethe design as described in U.S. Pate.No. 7,446,190, comprising anintracellular domain of CD3 chain (such as amino acid residues 52-163 ofthe human CD3 zeta chain, as shown in SEQ ID NO: 14 of U.S. Pat. No.7,446,190), a signaling region from CD28 and an antigen-binding element(or portion or domain; such as scFv). The CD28 portion, when between thezeta chain portion and the antigen-binding element, may suitably includethe transmembrane and signaling domains of CD28 (such as amino acidresidues 114-220 of SEQ ID NO: 10, full sequence shown in SEQ ID NO: 6of U.S. Pat. No. 7,446,190; these can include the following portion ofCD28 as set forth in Genbank identifier NM_006139 (sequence version 1, 2or 3): IEVMYPPPYLDNEKSNGTIIHVKGKHLCP SPLFP GP SKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS)) (SEQ. I.D. No. 27).Alternatively, when the zeta sequence lies between the CD28 sequence andthe antigen-binding element, intracellular domain of CD28 can be usedalone (such as amino sequence set forth in SEQ ID NO: 9 of U.S. Pat. No.7,446,190). Hence, certain embodiments employ a CAR comprising (a) azeta chain portion comprising the intracellular domain of human CD3ζchain, (b) a costimulatory signaling region, and (c) an antigen-bindingelement (or portion or domain), wherein the costimulatory signalingregion comprises the amino acid sequence encoded by SEQ ID NO: 6 of U.S.Pat. No. 7,446,190.

Alternatively, costimulation may be orchestrated by expressing CARs inantigen-specific T cells, chosen so as to be activated and expandedfollowing engagement of their native αβTCR, for example by antigen onprofessional antigen-presenting cells, with attendant costimulation. Inaddition, additional engineered receptors may be provided on theimmunoresponsive cells, for example to improve targeting of a T-cellattack and/or minimize side effects

By means of an example and without limitation, Kochenderfer et al.,(2009) J Immunother. 32 (7): 689-702 described anti-CD19 chimericantigen receptors (CAR). FMC63-28Z CAR contained a single chain variableregion moiety (scFv) recognizing CD19 derived from the FMC63 mousehybridoma (described in Nicholson et al., (1997) Molecular Immunology34: 1157-1165), a portion of the human CD28 molecule, and theintracellular component of the human TCR-ζ molecule. FMC63-CD828BBZ CARcontained the FMC63 scFv, the hinge and transmembrane regions of the CD8molecule, the cytoplasmic portions of CD28 and 4-1BB, and thecytoplasmic component of the TCR-ζ molecule. The exact sequence of theCD28 molecule included in the FMC63-28Z CAR corresponded to Genbankidentifier NM_006139; the sequence included all amino acids startingwith the amino acid sequence IEVMYPPPY (SEQ. I.D. No. 28) and continuingall the way to the carboxy-terminus of the protein. To encode theanti-CD19 scFv component of the vector, the authors designed a DNAsequence which was based on a portion of a previously published CAR(Cooper et al., (2003) Blood 101: 1637-1644). This sequence encoded thefollowing components in frame from the 5′ end to the 3′ end: an XhoIsite, the human granulocyte-macrophage colony-stimulating factor(GM-CSF) receptor α-chain signal sequence, the FMC63 light chainvariable region (as in Nicholson et al., supra), a linker peptide (as inCooper et al., supra), the FMC63 heavy chain variable region (as inNicholson et al., supra), and a NotI site. A plasmid encoding thissequence was digested with XhoI and NotI. To form the MSGV-FMC63-28Zretroviral vector, the XhoI and NotI-digested fragment encoding theFMC63 scFv was ligated into a second XhoI and NotI-digested fragmentthat encoded the MSGV retroviral backbone (as in Hughes et al., (2005)Human Gene Therapy 16: 457-472) as well as part of the extracellularportion of human CD28, the entire transmembrane and cytoplasmic portionof human CD28, and the cytoplasmic portion of the human TCR-molecule (asin Maher et al., 2002) Nature Biotechnology 20: 70-75). The FMC63-28ZCAR is included in the KTE-C19 (axicabtagene ciloleucel) anti-CD19 CAR-Ttherapy product in development by Kite Pharma, Inc. for the treatment ofinter alia patients with relapsed/refractory aggressive B-cellnon-Hodgkin lymphoma (NHL). Accordingly, in certain embodiments, cellsintended for adoptive cell therapies, more particularly immunoresponsivecells such as T cells, may express the FMC63-28Z CAR as described byKochenderfer et al. (supra). Hence, in certain embodiments, cellsintended for adoptive cell therapies, more particularly immunoresponsivecells such as T cells, may comprise a CAR comprising an extracellularantigen-binding element (or portion or domain; such as scFv) thatspecifically binds to an antigen, an intracellular signaling domaincomprising an intracellular domain of a CD3ζ chain, and a costimulatorysignaling region comprising a signaling domain of CD28. Preferably, theCD28 amino acid sequence is as set forth in Genbank identifier NM_006139(sequence version 1,2 or 3) starting with the amino acid sequenceIEVMYPPPY (SEQ ID NO: 28) and continuing all the way to thecarboxy-terminus of the protein. The sequence is reproduced herein:IEVMYPPPYLDNEK SNGTIIHVKGKHLCP SPLFP GP SKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 29).Preferably, the antigen is CD19, more preferably the antigen-bindingelement is an anti-CD19 scFv, even more preferably the anti-CD19 scFv asdescribed by Kochenderfer et al. (supra).

Additional anti-CD19 CARs are further described in International PatentPublication No. WO2015187528. More particularly Example 1 and Table 1 ofWO2015187528, incorporated by reference herein, demonstrate thegeneration of anti-CD19 CARs based on a fully human anti-CD19 monoclonalantibody (47G4, as described in US20100104509) and murine anti-CD19monoclonal antibody (as described in Nicholson et al. and explainedabove). Various combinations of a signal sequence (human CD8-alpha orGM-CSF receptor), extracellular and transmembrane regions (humanCD8-alpha) and intracellular T-cell signalling domains (CD28-CD3ζ;4-1BB-CD3ζ; CD27-CD3ζ; CD28-CD27-CD3ζ, 4-1BB-CD27-CD3ζ; CD27-4-1BB-CD3ζ;CD28-CD27-FcεRI gamma chain; or CD28-FcεRI gamma chain) were disclosed.Hence, in certain embodiments, cells intended for adoptive celltherapies, more particularly immunoresponsive cells such as T cells, maycomprise a CAR comprising an extracellular antigen-binding element thatspecifically binds to an antigen, an extracellular and transmembraneregion as set forth in Table 1 of WO2015187528 and an intracellularT-cell signalling domain as set forth in Table 1 of WO2015187528.Preferably, the antigen is CD19, more preferably the antigen-bindingelement is an anti-CD19 scFv, even more preferably the mouse or humananti-CD19 scFv as described in Example 1 of WO2015187528. In certainembodiments, the CAR comprises, consists essentially of or consists ofan amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 asset forth in Table 1 of WO2015187528.

By means of an example and without limitation, chimeric antigen receptorthat recognizes the CD70 antigen is described in International PatentPublication No. WO2012058460A2 (see also, Park et al., CD70 as a targetfor chimeric antigen receptor T cells in head and neck squamous cellcarcinoma, Oral Oncol. 2018 March; 78:145-150; and Jin et al., CD70, anovel target of CAR T-cell therapy for gliomas, Neuro Oncol. 2018 Jan10; 20(1):55-65). CD70 is expressed by diffuse large B-cell andfollicular lymphoma and also by the malignant cells of Hodgkinslymphoma, Waldenstrom's macroglobulinemia and multiple myeloma, and byHTLV-1-and EBV-associated malignancies. (Agathanggelou et al.Am.J.Pathol. 1995; 147: 1152-1160; Hunter et al., Blood 2004; 104:4881.26; Lens et al., J Immunol. 2005; 174:6212-6219; Baba et al., J Virol.2008; 82:3843-3852.) In addition, CD70 is expressed by non-hematologicalmalignancies such as renal cell carcinoma and glioblastoma. (Junker etal., J Urol. 2005; 173:2150-2153; Chahlavi et al., Cancer Res 2005;65:5428-5438) Physiologically, CD70 expression is transient andrestricted to a subset of highly activated T, B, and dendritic cells.

By means of an example and without limitation, chimeric antigen receptorthat recognizes BCMA has been described (see, e.g., US20160046724A1;WO2016014789A2; WO2017211900A1; WO2015158671A1; US20180085444A1;WO2018028647A1; US20170283504A1; and WO2013154760A1).

In certain embodiments, the immune cell may, in addition to a CAR orexogenous TCR as described herein, further comprise a chimericinhibitory receptor (inhibitory CAR) that specifically binds to a secondtarget antigen and is capable of inducing an inhibitory orimmunosuppressive or repressive signal to the cell upon recognition ofthe second target antigen. In certain embodiments, the chimericinhibitory receptor comprises an extracellular antigen-binding element(or portion or domain) configured to specifically bind to a targetantigen, a transmembrane domain, and an intracellular immunosuppressiveor repressive signaling domain. In certain embodiments, the secondtarget antigen is an antigen that is not expressed on the surface of acancer cell or infected cell or the expression of which is downregulatedon a cancer cell or an infected cell. In certain embodiments, the secondtarget antigen is an MHC-class I molecule. In certain embodiments, theintracellular signaling domain comprises a functional signaling portionof an immune checkpoint molecule, such as for example PD-1 or CTLA4.Advantageously, the inclusion of such inhibitory CAR reduces the chanceof the engineered immune cells attacking non-target (e.g., non-cancer)tissues.

Alternatively, T-cells expressing CARs may be further modified to reduceor eliminate expression of endogenous TCRs in order to reduce off-targeteffects. Reduction or elimination of endogenous TCRs can reduceoff-target effects and increase the effectiveness of the T cells (U.S.Pat. No. 9,181,527). T cells stably lacking expression of a functionalTCR may be produced using a variety of approaches. T cells internalize,sort, and degrade the entire T cell receptor as a complex, with ahalf-life of about 10 hours in resting T cells and 3 hours in stimulatedT cells (von Essen, M. et al. 2004. J. Immunol. 173:384-393). Properfunctioning of the TCR complex requires the proper stoichiometric ratioof the proteins that compose the TCR complex. TCR function also requirestwo functioning TCR zeta proteins with ITAM motifs. The activation ofthe TCR upon engagement of its MHC-peptide ligand requires theengagement of several TCRs on the same T cell, which all must signalproperly. Thus, if a TCR complex is destabilized with proteins that donot associate properly or cannot signal optimally, the T cell will notbecome activated sufficiently to begin a cellular response.

Accordingly, in some embodiments, TCR expression may eliminated usingRNA interference (e.g., shRNA, siRNA, miRNA, etc.), CRISPR, or othermethods that target the nucleic acids encoding specific TCRs (e.g.,TCR-α and TCR-β) and/or CD3 chains in primary T cells. By blockingexpression of one or more of these proteins, the T cell will no longerproduce one or more of the key components of the TCR complex, therebydestabilizing the TCR complex and preventing cell surface expression ofa functional TCR.

In some instances, CAR may also comprise a switch mechanism forcontrolling expression and/or activation of the CAR. For example, a CARmay comprise an extracellular, transmembrane, and intracellular domain,in which the extracellular domain comprises a target-specific bindingelement that comprises a label, binding domain, or tag that is specificfor a molecule other than the target antigen that is expressed on or bya target cell. In such embodiments, the specificity of the CAR isprovided by a second construct that comprises a target antigen bindingdomain (e.g., an scFv or a bispecific antibody that is specific for boththe target antigen and the label or tag on the CAR) and a domain that isrecognized by or binds to the label, binding domain, or tag on the CAR.See, e.g., WO 2013/044225, WO 2016/000304, WO 2015/057834, WO2015/057852, WO 2016/070061, U.S. Pat. No. 9,233,125, US 2016/0129109.In this way, a T-cell that expresses the CAR can be administered to asubject, but the CAR cannot bind its target antigen until the secondcomposition comprising an antigen-specific binding domain isadministered.

Alternative switch mechanisms include CARs that require multimerizationin order to activate their signaling function (see, e.g., US2015/0368342, US 2016/0175359, US 2015/0368360) and/or an exogenoussignal, such as a small molecule drug (US 2016/0166613, Yung et al.,Science, 2015), in order to elicit a T-cell response. Some CARs may alsocomprise a “suicide switch” to induce cell death of the CAR T-cellsfollowing treatment (Buddee et al., PLoS One, 2013) or to downregulateexpression of the CAR following binding to the target antigen (WO2016/011210).

Alternative techniques may be used to transform target immunoresponsivecells, such as protoplast fusion, lipofection, transfection orelectroporation. A wide variety of vectors may be used, such asretroviral vectors, lentiviral vectors, adenoviral vectors,adeno-associated viral vectors, plasmids or transposons, such as aSleeping Beauty transposon (see U.S. Pat. Nos. 6,489,458; 7,148,203;7,160,682; 7,985,739; 8,227,432), may be used to introduce CARs, forexample using 2nd generation antigen-specific CARs signaling throughCD3t and either CD28 or CD137. Viral vectors may for example includevectors based on HIV, SV40, EBV, HSV or BPV.

Cells that are targeted for transformation may for example include Tcells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL),regulatory T cells, human embryonic stem cells, tumor-infiltratinglymphocytes (TIL) or a pluripotent stem cell from which lymphoid cellsmay be differentiated. T cells expressing a desired CAR may for examplebe selected through co-culture with γ-irradiated activating andpropagating cells (AaPC), which co-express the cancer antigen andco-stimulatory molecules. The engineered CAR T-cells may be expanded,for example by co-culture on AaPC in presence of soluble factors, suchas IL-2 and IL-21. This expansion may for example be carried out so asto provide memory CAR+ T cells (which may for example be assayed bynon-enzymatic digital array and/or multi-panel flow cytometry). In thisway, CAR T cells may be provided that have specific cytotoxic activityagainst antigen-bearing tumors (optionally in conjunction withproduction of desired chemokines such as interferon-γ). CAR T cells ofthis kind may for example be used in animal models, for example to treattumor xenografts.

In certain embodiments, ACT includes co-transferring CD4+ TH1 cells andCD8+ CTLs to induce a synergistic antitumour response (see, e.g., Li etal., Adoptive cell therapy with CD4+ T helper 1 cells and CD8+ cytotoxicT cells enhances complete rejection of an established tumour, leading togeneration of endogenous memory responses to non-targeted tumourepitopes. Clin Transl Immunology. 2017 October; 6(10): e160).

In certain embodiments, Th17 cells are transferred to a subject in needthereof. Th17 cells have been reported to directly eradicate melanomatumors in mice to a greater extent than TH1 cells (Muranski P, et al.,Tumor-specific Th17-polarized cells eradicate large establishedmelanoma. Blood. 2008 Jul. 15; 112(2):362-73; and Martin-Orozco N, etal., T helper 17 cells promote cytotoxic T cell activation in tumorimmunity. Immunity. 2009 Nov. 20; 31(5):787-98). Those studies involvedan adoptive T cell transfer (ACT) therapy approach, which takesadvantage of CD4⁺ T cells that express a TCR recognizing tyrosinasetumor antigen. Exploitation of the TCR leads to rapid expansion of Th17populations to large numbers ex vivo for reinfusion into the autologoustumor-bearing hosts.

In certain embodiments, ACT may include autologous iPSC-based vaccines,such as irradiated iPSCs in autologous anti-tumor vaccines (see e.g.,Kooreman, Nigel G. et al., Autologous iPSC-Based Vaccines ElicitAnti-tumor Responses In vivo, Cell Stem Cell 22,1-13, 2018,doi.org/10.1016/j.stem.2018.01.016).

Unlike T-cell receptors (TCRs) that are MHC restricted, CARs canpotentially bind any cell surface-expressed antigen and can thus be moreuniversally used to treat patients (see Irving et al., EngineeringChimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don'tForget the Fuel, Front. Immunol., 3 Apr. 2017,doi.org/10.3389/fimmu.2017.00267). In certain embodiments, in theabsence of endogenous T-cell infiltrate (e.g., due to aberrant antigenprocessing and presentation), which precludes the use of TIL therapy andimmune checkpoint blockade, the transfer of CAR T-cells may be used totreat patients (see, e.g., Hinrichs C S, Rosenberg S A. Exploiting thecurative potential of adoptive T-cell therapy for cancer. Immunol Rev(2014) 257(1):56-71. doi:10.1111/ imr.12132).

Approaches such as the foregoing may be adapted to provide methods oftreating and/or increasing survival of a subject having a disease, suchas a neoplasia, for example by administering an effective amount of animmunoresponsive cell comprising an antigen recognizing receptor thatbinds a selected antigen, wherein the binding activates theimmunoresponsive cell, thereby treating or preventing the disease (suchas a neoplasia, a pathogen infection, an autoimmune disorder, or anallogeneic transplant reaction).

In certain embodiments, the treatment can be administered afterlymphodepleting pretreatment in the form of chemotherapy (typically acombination of cyclophosphamide and fludarabine) or radiation therapy.Initial studies in ACT had short lived responses and the transferredcells did not persist in vivo for very long (Houot et al., T-cell-basedimmunotherapy: adoptive cell transfer and checkpoint inhibition. CancerImmunol Res (2015) 3(10):1115-22; and Kamta et al., Advancing CancerTherapy with Present and Emerging Immuno-Oncology Approaches. Front.Oncol. (2017) 7:64). Immune suppressor cells like Tregs and MDSCs mayattenuate the activity of transferred cells by outcompeting them for thenecessary cytokines. Not being bound by a theory lymphodepletingpretreatment may eliminate the suppressor cells allowing the TILs topersist.

In one embodiment, the treatment can be administrated into patientsundergoing an immunosuppressive treatment (e.g., glucocorticoidtreatment). The cells or population of cells, may be made resistant toat least one immunosuppressive agent due to the inactivation of a geneencoding a receptor for such immunosuppressive agent. In certainembodiments, the immunosuppressive treatment provides for the selectionand expansion of the immunoresponsive T cells within the patient.

In certain embodiments, the treatment can be administered before primarytreatment (e.g., surgery or radiation therapy) to shrink a tumor beforethe primary treatment. In another embodiment, the treatment can beadministered after primary treatment to remove any remaining cancercells.

In certain embodiments, immunometabolic barriers can be targetedtherapeutically prior to and/or during ACT to enhance responses to ACTor CAR T-cell therapy and to support endogenous immunity (see, e.g.,Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racingin Solid Tumors: Don't Forget the Fuel, Front. Immunol., 3 Apr. 2017,doi.org/10.3389/fimmu.2017.00267).

The administration of cells or population of cells, such as immunesystem cells or cell populations, such as more particularlyimmunoresponsive cells or cell populations, as disclosed herein may becarried out in any convenient manner, including by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation. Thecells or population of cells may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, intrathecally, by intravenous orintralymphatic injection, or intraperitoneally. In some embodiments, thedisclosed CARs may be delivered or administered into a cavity formed bythe resection of tumor tissue (i.e. intracavity delivery) or directlyinto a tumor prior to resection (i.e. intratumoral delivery). In oneembodiment, the cell compositions of the present invention arepreferably administered by intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 10⁴-10⁹ cells per kg body weight, preferably 10⁵to 10⁶ cells/kg body weight including all integer values of cell numberswithin those ranges. Dosing in CAR T cell therapies may for exampleinvolve administration of from 10⁶ to 10⁹ cells/kg, with or without acourse of lymphodepletion, for example with cyclophosphamide. The cellsor population of cells can be administrated in one or more doses. Inanother embodiment, the effective amount of cells are administrated as asingle dose. In another embodiment, the effective amount of cells areadministrated as more than one dose over a period time. Timing ofadministration is within the judgment of managing physician and dependson the clinical condition of the patient. The cells or population ofcells may be obtained from any source, such as a blood bank or a donor.While individual needs vary, determination of optimal ranges ofeffective amounts of a given cell type for a particular disease orconditions are within the skill of one in the art. An effective amountmeans an amount which provides a therapeutic or prophylactic benefit.The dosage administrated will be dependent upon the age, health andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment and the nature of the effect desired.

In another embodiment, the effective amount of cells or compositioncomprising those cells are administrated parenterally. Theadministration can be an intravenous administration. The administrationcan be directly done by injection within a tumor.

To guard against possible adverse reactions, engineered immunoresponsivecells may be equipped with a transgenic safety switch, in the form of atransgene that renders the cells vulnerable to exposure to a specificsignal. For example, the herpes simplex viral thymidine kinase (TK) genemay be used in this way, for example by introduction into allogeneic Tlymphocytes used as donor lymphocyte infusions following stem celltransplantation (Greco, et al., Improving the safety of cell therapywith the TK-suicide gene. Front. Pharmacol. 2015; 6: 95). In such cells,administration of a nucleoside prodrug such as ganciclovir or acyclovircauses cell death. Alternative safety switch constructs includeinducible caspase 9, for example triggered by administration of asmall-molecule dimerizer that brings together two nonfunctional icasp9molecules to form the active enzyme. A wide variety of alternativeapproaches to implementing cellular proliferation controls have beendescribed (see U.S. Patent Publication No. 20130071414; PCT PatentPublication WO2011146862; PCT Patent Publication WO2014011987; PCTPatent Publication WO2013040371; Zhou et al. BLOOD, 2014,123/25:3895-3905; Di Stasi et al., The New England Journal of Medicine2011; 365:1673-1683; Sadelain M, The New England Journal of Medicine2011; 365:1735-173; Ramos et al., Stem Cells 28(6):1107-15 (2010)).

In a further refinement of adoptive therapies, genome editing may beused to tailor immunoresponsive cells to alternative implementations,for example providing edited CAR T cells (see Poirot et al., 2015,Multiplex genome edited T-cell manufacturing platform for“off-the-shelf” adoptive T-cell immunotherapies, Cancer Res 75 (18):3853; Ren et al., 2017, Multiplex genome editing to generate universalCAR T cells resistant to PD1 inhibition, Clin Cancer Res. 2017 May 1;23(9):2255-2266. doi: 10.1158/1078-0432.CCR-16-1300. Epub 2016 Nov 4;Qasim et al., 2017, Molecular remission of infant B-ALL after infusionof universal TALEN gene-edited CART cells, Sci Transl Med. 2017 Jan. 25;9(374); Legut, et al., 2018, CRISPR-mediated TCR replacement generatessuperior anticancer transgenic T cells. Blood, 131(3), 311-322; andGeorgiadis et al., Long Terminal Repeat CRISPR-CAR-Coupled “Universal” TCells Mediate Potent Anti-leukemic Effects, Molecular Therapy, In Press,Corrected Proof, Available online 6 Mar. 2018). Cells may be editedusing any CRISPR system and method of use thereof as described herein.CRISPR systems may be delivered to an immune cell by any methoddescribed herein. In preferred embodiments, cells are edited ex vivo andtransferred to a subject in need thereof. Immunoresponsive cells, CAR Tcells or any cells used for adoptive cell transfer may be edited.Editing may be performed for example to insert or knock-in an exogenousgene, such as an exogenous gene encoding a CAR or a TCR, at apreselected locus in a cell (e.g. TRAC locus); to eliminate potentialalloreactive T-cell receptors (TCR) or to prevent inappropriate pairingbetween endogenous and exogenous TCR chains, such as to knock-out orknock-down expression of an endogenous TCR in a cell; to disrupt thetarget of a chemotherapeutic agent in a cell; to block an immunecheckpoint, such as to knock-out or knock-down expression of an immunecheckpoint protein or receptor in a cell; to knock-out or knock-downexpression of other gene or genes in a cell, the reduced expression orlack of expression of which can enhance the efficacy of adoptivetherapies using the cell; to knock-out or knock-down expression of anendogenous gene in a cell, said endogenous gene encoding an antigentargeted by an exogenous CAR or TCR; to knock-out or knock-downexpression of one or more WIC constituent proteins in a cell; toactivate a T cell; to modulate cells such that the cells are resistantto exhaustion or dysfunction; and/or increase the differentiation and/orproliferation of functionally exhausted or dysfunctional CD8+ T-cells(see PCT Patent Publications: WO2013176915, WO2014059173, WO2014172606,WO2014184744, and WO2014191128).

In certain embodiments, editing may result in inactivation of a gene. Byinactivating a gene, it is intended that the gene of interest is notexpressed in a functional protein form. In a particular embodiment, theCRISPR system specifically catalyzes cleavage in one targeted genethereby inactivating said targeted gene. The nucleic acid strand breakscaused are commonly repaired through the distinct mechanisms ofhomologous recombination or non-homologous end joining (NHEJ). However,NHEJ is an imperfect repair process that often results in changes to theDNA sequence at the site of the cleavage. Repair via non-homologous endjoining (NHEJ) often results in small insertions or deletions (Indel)and can be used for the creation of specific gene knockouts. Cells inwhich a cleavage induced mutagenesis event has occurred can beidentified and/or selected by well-known methods in the art. In certainembodiments, homology directed repair (HDR) is used to concurrentlyinactivate a gene (e.g., TRAC) and insert an endogenous TCR or CAR intothe inactivated locus.

Hence, in certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toinsert or knock-in an exogenous gene, such as an exogenous gene encodinga CAR or a TCR, at a preselected locus in a cell. Conventionally,nucleic acid molecules encoding CARs or TCRs are transfected ortransduced to cells using randomly integrating vectors, which, dependingon the site of integration, may lead to clonal expansion, oncogenictransformation, variegated transgene expression and/or transcriptionalsilencing of the transgene. Directing of transgene(s) to a specificlocus in a cell can minimize or avoid such risks and advantageouslyprovide for uniform expression of the transgene(s) by the cells. Withoutlimitation, suitable ‘safe harbor’ loci for directed transgeneintegration include CCR5 or AAVS1. Homology-directed repair (HDR)strategies are known and described elsewhere in this specificationallowing to insert transgenes into desired loci (e.g., TRAC locus).

Further suitable loci for insertion of transgenes, in particular CAR orexogenous TCR transgenes, include without limitation loci comprisinggenes coding for constituents of endogenous T-cell receptor, such asT-cell receptor alpha locus (TRA) or T-cell receptor beta locus (TRB),for example T-cell receptor alpha constant (TRAC) locus, T-cell receptorbeta constant 1 (TRBC1) locus or T-cell receptor beta constant 2 (TRBC1)locus. Advantageously, insertion of a transgene into such locus cansimultaneously achieve expression of the transgene, potentiallycontrolled by the endogenous promoter, and knock-out expression of theendogenous TCR. This approach has been exemplified in Eyquem et al.,(2017) Nature 543: 113-117, wherein the authors used CRISPR/Cas9 geneediting to knock-in a DNA molecule encoding a CD19-specific CAR into theTRAC locus downstream of the endogenous promoter; the CAR-T cellsobtained by CRISPR were significantly superior in terms of reduced tonicCAR signaling and exhaustion.

T cell receptors (TCR) are cell surface receptors that participate inthe activation of T cells in response to the presentation of antigen.The TCR is generally made from two chains, a and β, which assemble toform a heterodimer and associates with the CD3-transducing subunits toform the T cell receptor complex present on the cell surface. Each α andβ chain of the TCR consists of an immunoglobulin-like N-terminalvariable (V) and constant (C) region, a hydrophobic transmembranedomain, and a short cytoplasmic region. As for immunoglobulin molecules,the variable region of the α and β chains are generated by V(D)Jrecombination, creating a large diversity of antigen specificitieswithin the population of T cells. However, in contrast toimmunoglobulins that recognize intact antigen, T cells are activated byprocessed peptide fragments in association with an MHC molecule,introducing an extra dimension to antigen recognition by T cells, knownas MHC restriction. Recognition of MHC disparities between the donor andrecipient through the T cell receptor leads to T cell proliferation andthe potential development of graft versus host disease (GVHD). Theinactivation of TCRα or TCRβ can result in the elimination of the TCRfrom the surface of T cells preventing recognition of alloantigen andthus GVHD. However, TCR disruption generally results in the eliminationof the CD3 signaling component and alters the means of further T cellexpansion.

Hence, in certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toknock-out or knock-down expression of an endogenous TCR in a cell. Forexample, NHEJ-based or HDR-based gene editing approaches can be employedto disrupt the endogenous TCR alpha and/or beta chain genes. Forexample, gene editing system or systems, such as CRISPR/Cas system orsystems, can be designed to target a sequence found within the TCR betachain conserved between the beta 1 and beta 2 constant region genes(TRBC1 and TRBC2) and/or to target the constant region of the TCR alphachain (TRAC) gene.

Allogeneic cells are rapidly rejected by the host immune system. It hasbeen demonstrated that, allogeneic leukocytes present in non-irradiatedblood products will persist for no more than 5 to 6 days (Boni, Muranskiet al. 2008 Blood 1; 112(12):4746-54). Thus, to prevent rejection ofallogeneic cells, the host's immune system usually has to be suppressedto some extent. However, in the case of adoptive cell transfer the useof immunosuppressive drugs also have a detrimental effect on theintroduced therapeutic T cells. Therefore, to effectively use anadoptive immunotherapy approach in these conditions, the introducedcells would need to be resistant to the immunosuppressive treatment.Thus, in a particular embodiment, the present invention furthercomprises a step of modifying T cells to make them resistant to animmunosuppressive agent, preferably by inactivating at least one geneencoding a target for an immunosuppressive agent. An immunosuppressiveagent is an agent that suppresses immune function by one of severalmechanisms of action. An immunosuppressive agent can be, but is notlimited to a calcineurin inhibitor, a target of rapamycin, aninterleukin-2 receptor α-chain blocker, an inhibitor of inosinemonophosphate dehydrogenase, an inhibitor of dihydrofolic acidreductase, a corticosteroid or an immunosuppressive antimetabolite. Thepresent invention allows conferring immunosuppressive resistance to Tcells for immunotherapy by inactivating the target of theimmunosuppressive agent in T cells. As non-limiting examples, targetsfor an immunosuppressive agent can be a receptor for animmunosuppressive agent such as: CD52, glucocorticoid receptor (GR), aFKBP family gene member and a cyclophilin family gene member.

In certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toblock an immune checkpoint, such as to knock-out or knock-downexpression of an immune checkpoint protein or receptor in a cell. Immunecheckpoints are inhibitory pathways that slow down or stop immunereactions and prevent excessive tissue damage from uncontrolled activityof immune cells. In certain embodiments, the immune checkpoint targetedis the programmed death-1 (PD-1 or CD279) gene (PDCD1). In otherembodiments, the immune checkpoint targeted is cytotoxicT-lymphocyte-associated antigen (CTLA-4). In additional embodiments, theimmune checkpoint targeted is another member of the CD28 and CTLA4 Igsuperfamily such as BTLA, LAG3, ICOS, PDL1 or KIR. In further additionalembodiments, the immune checkpoint targeted is a member of the TNFRsuperfamily such as CD40, OX40, CD137, GITR, CD27 or TIM-3.

Additional immune checkpoints include Src homology 2 domain-containingprotein tyrosine phosphatase 1 (SHP-1) (Watson H A, et al., SHP-1: thenext checkpoint target for cancer immunotherapy? Biochem Soc Trans. 2016Apr. 15; 44(2):356-62). SHP-1 is a widely expressed inhibitory proteintyrosine phosphatase (PTP). In T-cells, it is a negative regulator ofantigen-dependent activation and proliferation. It is a cytosolicprotein, and therefore not amenable to antibody-mediated therapies, butits role in activation and proliferation makes it an attractive targetfor genetic manipulation in adoptive transfer strategies, such aschimeric antigen receptor (CAR) T cells. Immune checkpoints may alsoinclude T cell immunoreceptor with Ig and ITIM domains(TIGIT/Vstm3/WUCAM/VSIG9) and VISTA (Le Mercier I, et al., (2015) BeyondCTLA-4 and PD-1, the generation Z of negative checkpoint regulators.Front. Immunol. 6:418).

International Patent Publication No. WO2014172606 relates to the use ofMT1 and/or MT2 inhibitors to increase proliferation and/or activity ofexhausted CD8+ T-cells and to decrease CD8+ T-cell exhaustion (e.g.,decrease functionally exhausted or unresponsive CD8+ immune cells). Incertain embodiments, metallothioneins are targeted by gene editing inadoptively transferred T cells.

In certain embodiments, targets of gene editing may be at least onetargeted locus involved in the expression of an immune checkpointprotein. Such targets may include, but are not limited to CTLA4, PPP2CA,PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2,BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4),TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS,TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA,IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SITZ, FOXP3, PRDM1,BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, MT1, MT2, CD40, OX40,CD137, GITR, CD27, SHP-1, TIM-3, CEACAM-1, CEACAM-3, or CEACAM-5. Inpreferred embodiments, the gene locus involved in the expression of PD-1or CTLA-4 genes is targeted. In other preferred embodiments,combinations of genes are targeted, such as but not limited to PD-1 andTIGIT.

By means of an example and without limitation, International PatentPublication No. WO2016196388 concerns an engineered T cell comprising(a) a genetically engineered antigen receptor that specifically binds toan antigen, which receptor may be a CAR; and (b) a disrupted geneencoding a PD-L1, an agent for disruption of a gene encoding a PD-L1,and/or disruption of a gene encoding PD-L1, wherein the disruption ofthe gene may be mediated by a gene editing nuclease, a zinc fingernuclease (ZFN), CRISPR/Cas9 and/or TALEN. WO2015142675 relates to immuneeffector cells comprising a CAR in combination with an agent (such asCRISPR, TALEN or ZFN) that increases the efficacy of the immune effectorcells in the treatment of cancer, wherein the agent may inhibit animmune inhibitory molecule, such as PD1, PD-L1, CTLA-4, TIM-3, LAG-3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, orCEACAM-5. Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266 performedlentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAstargeting endogenous TCR, (3-2 microglobulin (B2M) and PD1simultaneously, to generate gene-disrupted allogeneic CAR T cellsdeficient of TCR, HLA class I molecule and PD1.

In certain embodiments, cells may be engineered to express a CAR,wherein expression and/or function of methylcytosine dioxygenase genes(TET1, TET2 and/or TET3) in the cells has been reduced or eliminated,such as by CRISPR, ZNF or TALEN (for example, as described inWO201704916).

In certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toknock-out or knock-down expression of an endogenous gene in a cell, saidendogenous gene encoding an antigen targeted by an exogenous CAR or TCR,thereby reducing the likelihood of targeting of the engineered cells. Incertain embodiments, the targeted antigen may be one or more antigenselected from the group consisting of CD38, CD138, CS-1, CD33, CD26,CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, CD362, humantelomerase reverse transcriptase (hTERT), survivin, mouse double minute2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumorgene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen(CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen(PSMA), p53, cyclin (D1), B cell maturation antigen (BCMA),transmembrane activator and CAML Interactor (TACI), and B-cellactivating factor receptor (BAFF-R) (for example, as described inInternational Patent Publication Nos. WO2016011210 and WO2017011804).

In certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toknock-out or knock-down expression of one or more MHC constituentproteins, such as one or more HLA proteins and/or beta-2 microglobulin(B2M), in a cell, whereby rejection of non-autologous (e.g., allogeneic)cells by the recipient's immune system can be reduced or avoided. Inpreferred embodiments, one or more HLA class I proteins, such as HLA-A,B and/or C, and/or B2M may be knocked-out or knocked-down. Preferably,B2M may be knocked-out or knocked-down. By means of an example, Ren etal., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviraldelivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targetingendogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, togenerate gene-disrupted allogeneic CAR T cells deficient of TCR, HLAclass I molecule and PD1.

In other embodiments, at least two genes are edited. Pairs of genes mayinclude, but are not limited to PD1 and TCRα, PD1 and TCRβ, CTLA-4 andTCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ,TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 andTCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 andTCRα, 2B4 and TCRβ, B2M and TCRα, B2M and TCRβ.

In certain embodiments, a cell may be multiply edited (multiplex genomeediting) as taught herein to (1) knock-out or knock-down expression ofan endogenous TCR (for example, TRBC1, TRBC2 and/or TRAC), (2) knock-outor knock-down expression of an immune checkpoint protein or receptor(for example PD1, PD-L1 and/or CTLA4); and (3) knock-out or knock-downexpression of one or more MHC constituent proteins (for example, HLA-A,B and/or C, and/or B2M, preferably B2M).

Whether prior to or after genetic modification of the T cells, the Tcells can be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566;7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631. Tcells can be expanded in vitro or in vivo.

Immune cells may be obtained using any method known in the art. In oneembodiment, allogenic T cells may be obtained from healthy subjects. Inone embodiment T cells that have infiltrated a tumor are isolated. Tcells may be removed during surgery. T cells may be isolated afterremoval of tumor tissue by biopsy. T cells may be isolated by any meansknown in the art. In one embodiment, T cells are obtained by apheresis.In one embodiment, the method may comprise obtaining a bulk populationof T cells from a tumor sample by any suitable method known in the art.For example, a bulk population of T cells can be obtained from a tumorsample by dissociating the tumor sample into a cell suspension fromwhich specific cell populations can be selected. Suitable methods ofobtaining a bulk population of T cells may include, but are not limitedto, any one or more of mechanically dissociating (e.g., mincing) thetumor, enzymatically dissociating (e.g., digesting) the tumor, andaspiration (e.g., as with a needle).

The bulk population of T cells obtained from a tumor sample may compriseany suitable type of T cell. Preferably, the bulk population of T cellsobtained from a tumor sample comprises tumor infiltrating lymphocytes(TILs).

The tumor sample may be obtained from any mammal. Unless statedotherwise, as used herein, the term “mammal” refers to any mammalincluding, but not limited to, mammals of the order Logomorpha, such asrabbits; the order Carnivora, including Felines (cats) and Canines(dogs); the order Artiodactyla, including Bovines (cows) and Swines(pigs); or of the order Perssodactyla, including Equines (horses). Themammals may be non-human primates, e.g., of the order Primates, Ceboids,or Simoids (monkeys) or of the order Anthropoids (humans and apes). Insome embodiments, the mammal may be a mammal of the order Rodentia, suchas mice and hamsters. Preferably, the mammal is a non-human primate or ahuman. An especially preferred mammal is the human.

T cells can be obtained from a number of sources, including peripheralblood mononuclear cells (PBMC), bone marrow, lymph node tissue, spleentissue, and tumors. In certain embodiments of the present invention, Tcells can be obtained from a unit of blood collected from a subjectusing any number of techniques known to the skilled artisan, such asFicoll separation. In one preferred embodiment, cells from thecirculating blood of an individual are obtained by apheresis orleukapheresis. The apheresis product typically contains lymphocytes,including T cells, monocytes, granulocytes, B cells, other nucleatedwhite blood cells, red blood cells, and platelets. In one embodiment,the cells collected by apheresis may be washed to remove the plasmafraction and to place the cells in an appropriate buffer or media forsubsequent processing steps. In one embodiment of the invention, thecells are washed with phosphate buffered saline (PBS). In an alternativeembodiment, the wash solution lacks calcium and may lack magnesium ormay lack many if not all divalent cations. Initial activation steps inthe absence of calcium lead to magnified activation. As those ofordinary skill in the art would readily appreciate a washing step may beaccomplished by methods known to those in the art, such as by using asemi-automated “flow-through” centrifuge (for example, the Cobe 2991cell processor) according to the manufacturer's instructions. Afterwashing, the cells may be resuspended in a variety of biocompatiblebuffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theundesirable components of the apheresis sample may be removed and thecells directly resuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient. A specificsubpopulation of T cells, such as CD28+, CD4+, CDC, CD45RA+, andCD45RO+T cells, can be further isolated by positive or negativeselection techniques. For example, in one preferred embodiment, T cellsare isolated by incubation with anti-CD3/anti-CD28 (i.e.,3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, or XCYTEDYNABEADS™ for a time period sufficient for positive selection of thedesired T cells. In one embodiment, the time period is about 30 minutes.In a further embodiment, the time period ranges from 30 minutes to 36hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another preferred embodiment, the time period is 10 to 24 hours. Inone preferred embodiment, the incubation time period is 24 hours. Forisolation of T cells from patients with leukemia, use of longerincubation times, such as 24 hours, can increase cell yield. Longerincubation times may be used to isolate T cells in any situation wherethere are few T cells as compared to other cell types, such in isolatingtumor infiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. A preferred method iscell sorting and/or selection via negative magnetic immunoadherence orflow cytometry that uses a cocktail of monoclonal antibodies directed tocell surface markers present on the cells negatively selected. Forexample, to enrich for CD4+ cells by negative selection, a monoclonalantibody cocktail typically includes antibodies to CD14, CD20, CD11b,CD16, HLA-DR, and CD8.

Further, monocyte populations (i.e., CD14+ cells) may be depleted fromblood preparations by a variety of methodologies, including anti-CD14coated beads or columns, or utilization of the phagocytotic activity ofthese cells to facilitate removal. Accordingly, in one embodiment, theinvention uses paramagnetic particles of a size sufficient to beengulfed by phagocytotic monocytes. In certain embodiments, theparamagnetic particles are commercially available beads, for example,those produced by Life Technologies under the trade name Dynabeads™. Inone embodiment, other non-specific cells are removed by coating theparamagnetic particles with “irrelevant” proteins (e.g., serum proteinsor antibodies). Irrelevant proteins and antibodies include thoseproteins and antibodies or fragments thereof that do not specificallytarget the T cells to be isolated. In certain embodiments, theirrelevant beads include beads coated with sheep anti-mouse antibodies,goat anti-mouse antibodies, and human serum albumin.

In brief, such depletion of monocytes is performed by preincubating Tcells isolated from whole blood, apheresed peripheral blood, or tumorswith one or more varieties of irrelevant or non-antibody coupledparamagnetic particles at any amount that allows for removal ofmonocytes (approximately a 20:1 bead:cell ratio) for about 30 minutes to2 hours at 22 to 37 degrees C., followed by magnetic removal of cellswhich have attached to or engulfed the paramagnetic particles. Suchseparation can be performed using standard methods available in the art.For example, any magnetic separation methodology may be used including avariety of which are commercially available, (e.g., DYNAL® MagneticParticle Concentrator (DYNAL MPC®)). Assurance of requisite depletioncan be monitored by a variety of methodologies known to those ofordinary skill in the art, including flow cytometric analysis of CD14positive cells, before and after depletion.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8+ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

T cells can also be frozen. Wishing not to be bound by theory, thefreeze and subsequent thaw step provides a more uniform product byremoving granulocytes and to some extent monocytes in the cellpopulation. After a washing step to remove plasma and platelets, thecells may be suspended in a freezing solution. While many freezingsolutions and parameters are known in the art and will be useful in thiscontext, one method involves using PBS containing 20% DMSO and 8% humanserum albumin, or other suitable cell freezing media, the cells then arefrozen to −80° C. at a rate of 1° per minute and stored in the vaporphase of a liquid nitrogen storage tank. Other methods of controlledfreezing may be used as well as uncontrolled freezing immediately at−20° C. or in liquid nitrogen.

T cells for use in the present invention may also be antigen-specific Tcells. For example, tumor-specific T cells can be used. In certainembodiments, antigen-specific T cells can be isolated from a patient ofinterest, such as a patient afflicted with a cancer or an infectiousdisease. In one embodiment, neoepitopes are determined for a subject andT cells specific to these antigens are isolated. Antigen-specific cellsfor use in expansion may also be generated in vitro using any number ofmethods known in the art, for example, as described in U.S. PatentPublication No. US 20040224402 entitled, Generation and Isolation ofAntigen-Specific T Cells, or in U.S. Pat. No. 6,040,177.Antigen-specific cells for use in the present invention may also begenerated using any number of methods known in the art, for example, asdescribed in Current Protocols in Immunology, or Current Protocols inCell Biology, both published by John Wiley & Sons, Inc., Boston, Mass.

In a related embodiment, it may be desirable to sort or otherwisepositively select (e.g. via magnetic selection) the antigen specificcells prior to or following one or two rounds of expansion. Sorting orpositively selecting antigen-specific cells can be carried out usingpeptide-MEW tetramers (Altman, et al., Science. 1996 Oct. 4;274(5284):94-6). In another embodiment, the adaptable tetramertechnology approach is used (Andersen et al., 2012 Nat Protoc.7:891-902). Tetramers are limited by the need to utilize predictedbinding peptides based on prior hypotheses, and the restriction tospecific HLAs. Peptide-MHC tetramers can be generated using techniquesknown in the art and can be made with any MEW molecule of interest andany antigen of interest as described herein. Specific epitopes to beused in this context can be identified using numerous assays known inthe art. For example, the ability of a polypeptide to bind to MEW classI may be evaluated indirectly by monitoring the ability to promoteincorporation of ¹²⁵I labeled β2-microglobulin (β2m) into MHC classI/β2m/peptide heterotrimeric complexes (see Parker et al., J. Immunol.152:163, 1994).

In one embodiment cells are directly labeled with an epitope-specificreagent for isolation by flow cytometry followed by characterization ofphenotype and TCRs. In one embodiment, T cells are isolated bycontacting with T cell specific antibodies. Sorting of antigen-specificT cells, or generally any cells of the present invention, can be carriedout using any of a variety of commercially available cell sorters,including, but not limited to, MoFlo sorter (DakoCytomation, FortCollins, Colo.), FACSAria™, FACSArray™, FACSVantage™, BD™ LSR II, andFACSCalibur™ (BD Biosciences, San Jose, Calif.).

In a preferred embodiment, the method comprises selecting cells thatalso express CD3. The method may comprise specifically selecting thecells in any suitable manner. Preferably, the selecting is carried outusing flow cytometry. The flow cytometry may be carried out using anysuitable method known in the art. The flow cytometry may employ anysuitable antibodies and stains. Preferably, the antibody is chosen suchthat it specifically recognizes and binds to the particular biomarkerbeing selected. For example, the specific selection of CD3, CD8, TIM-3,LAG-3, 4-1BB, or PD-1 may be carried out using anti-CD3, anti-CD8,anti-TIM-3, anti-LAG-3, anti-4-1BB, or anti-PD-1 antibodies,respectively. The antibody or antibodies may be conjugated to a bead(e.g., a magnetic bead) or to a fluorochrome. Preferably, the flowcytometry is fluorescence-activated cell sorting (FACS). TCRs expressedon T cells can be selected based on reactivity to autologous tumors.Additionally, T cells that are reactive to tumors can be selected forbased on markers using the methods described in patent publication Nos.WO2014133567 and WO2014133568, herein incorporated by reference in theirentirety. Additionally, activated T cells can be selected for based onsurface expression of CD107a.

In one embodiment of the invention, the method further comprisesexpanding the numbers of T cells in the enriched cell population. Suchmethods are described in U.S. Pat. No. 8,637,307 and is hereinincorporated by reference in its entirety. The numbers of T cells may beincreased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), morepreferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-,or 90-fold), more preferably at least about 100-fold, more preferably atleast about 1,000 fold, or most preferably at least about 100,000-fold.The numbers of T cells may be expanded using any suitable method knownin the art. Exemplary methods of expanding the numbers of cells aredescribed in patent publication No. WO 2003057171, U.S. Pat. No.8,034,334, and U.S. Patent Application Publication No. 2012/0244133,each of which is incorporated herein by reference.

In one embodiment, ex vivo T cell expansion can be performed byisolation of T cells and subsequent stimulation or activation followedby further expansion. In one embodiment of the invention, the T cellsmay be stimulated or activated by a single agent. In another embodiment,T cells are stimulated or activated with two agents, one that induces aprimary signal and a second that is a co-stimulatory signal. Ligandsuseful for stimulating a single signal or stimulating a primary signaland an accessory molecule that stimulates a second signal may be used insoluble form. Ligands may be attached to the surface of a cell, to anEngineered Multivalent Signaling Platform (EMSP), or immobilized on asurface. In a preferred embodiment both primary and secondary agents areco-immobilized on a surface, for example a bead or a cell. In oneembodiment, the molecule providing the primary activation signal may bea CD3 ligand, and the co-stimulatory molecule may be a CD28 ligand or4-1BB ligand.

In certain embodiments, T cells comprising a CAR or an exogenous TCR,may be manufactured as described in International Patent Publication No.WO2015120096, by a method comprising enriching a population oflymphocytes obtained from a donor subject; stimulating the population oflymphocytes with one or more T-cell stimulating agents to produce apopulation of activated T cells, wherein the stimulation is performed ina closed system using serum-free culture medium; transducing thepopulation of activated T cells with a viral vector comprising a nucleicacid molecule which encodes the CAR or TCR, using a single cycletransduction to produce a population of transduced T cells, wherein thetransduction is performed in a closed system using serum-free culturemedium; and expanding the population of transduced T cells for apredetermined time to produce a population of engineered T cells,wherein the expansion is performed in a closed system using serum-freeculture medium. In certain embodiments, T cells comprising a CAR or anexogenous TCR, may be manufactured as described in WO2015120096, by amethod comprising: obtaining a population of lymphocytes; stimulatingthe population of lymphocytes with one or more stimulating agents toproduce a population of activated T cells, wherein the stimulation isperformed in a closed system using serum-free culture medium;transducing the population of activated T cells with a viral vectorcomprising a nucleic acid molecule which encodes the CAR or TCR, usingat least one cycle transduction to produce a population of transduced Tcells, wherein the transduction is performed in a closed system usingserum-free culture medium; and expanding the population of transduced Tcells to produce a population of engineered T cells, wherein theexpansion is performed in a closed system using serum-free culturemedium. The predetermined time for expanding the population oftransduced T cells may be 3 days. The time from enriching the populationof lymphocytes to producing the engineered T cells may be 6 days. Theclosed system may be a closed bag system. Further provided is populationof T cells comprising a CAR or an exogenous TCR obtainable or obtainedby said method, and a pharmaceutical composition comprising such cells.

In certain embodiments, T cell maturation or differentiation in vitromay be delayed or inhibited by the method as described in WO2017070395,comprising contacting one or more T cells from a subject in need of a Tcell therapy with an AKT inhibitor (such as, e.g., one or a combinationof two or more AKT inhibitors disclosed in claim 8 of WO2017070395) andat least one of exogenous Interleukin-7 (IL-7) and exogenousInterleukin-15 (IL-15), wherein the resulting T cells exhibit delayedmaturation or differentiation, and/or wherein the resulting T cellsexhibit improved T cell function (such as, e.g., increased T cellproliferation; increased cytokine production; and/or increased cytolyticactivity) relative to a T cell function of a T cell cultured in theabsence of an AKT inhibitor.

In certain embodiments, a patient in need of a T cell therapy may beconditioned by a method as described in WO2016191756 comprisingadministering to the patient a dose of cyclophosphamide between 200mg/m2/day and 2000 mg/m2/day and a dose of fludarabine between 20mg/m2/day and 900 mg/m²/day.

Methods of Enhancing an Anti-Cancer Immune Response

In another aspect, embodiments are directed to methods of enhancinganti-cancer immune responses by modulating one or more receptor-ligandinteractions identified in Table 1. In certain embodiments, modulatingan interaction modulates one or more functional T cell immune states,described further herein. In certain embodiments, a gene signature ismodified. For example, modulating one or more receptor-ligandinteractions alters a gene signature in the cells. The gene signaturemay be a pathway involved in an immune response.

In one example embodiment, a VEGFB-NRP1 interaction is inhibited by anantagonist to prevent VEGFB expressing tumors from interacting with NRP1expressing T cells. In one example embodiment, a CADM1-CRTAM interactionis inhibited by an antagonist to prevent CADM1 expressing tumors frominteracting with CRTAM expressing T cells. In certain embodiments, Tcell dysfunction is delayed or prevented by blocking communication withthe tumor.

In one example embodiment, a TFPI-SDC4 interaction is inhibited by anantagonist to prevent TFPI expressing tumors from interacting with SDC4expressing Treg cells. In one example embodiment, a MFGE8-ITGAVinteraction is inhibited by an antagonist to prevent MFGE8 expressingtumors from interacting with ITGAV expressing Treg cells. In one exampleembodiment, a ADAM12-SDC4 interaction is inhibited by an antagonist toprevent ADAM12 expressing tumors from interacting with SDC4 expressingTreg cells. In one example embodiment, a CYR61-ITGAV interaction isinhibited by an antagonist to prevent CYR61 expressing tumors frominteracting with ITGAV expressing Treg cells. In certain embodiments,Treg activation is delayed or prevented by blocking communication withthe tumor.

Functional T Cell Immune States

In certain embodiments, the present invention provides for modulatingimmune states by modifying one or more interacting cell types. Theimmune state can be modulated by modulating T cell activation, T celldysfunction or T cell regulatory function. In certain embodiments, Tcells can affect the overall immune state, such as other immune cells inproximity.

The term “modulate” broadly denotes a qualitative and/or quantitativealteration, change or variation in that which is being modulated. Wheremodulation can be assessed quantitatively—for example, where modulationcomprises or consists of a change in a quantifiable variable such as aquantifiable property of a cell or where a quantifiable variableprovides a suitable surrogate for the modulation—modulation specificallyencompasses both increase (e.g., activation) or decrease (e.g.,inhibition) in the measured variable. The term encompasses any extent ofsuch modulation, e.g., any extent of such increase or decrease, and maymore particularly refer to statistically significant increase ordecrease in the measured variable. By means of example, modulation mayencompass an increase in the value of the measured variable by at leastabout 10%, e.g., by at least about 20%, preferably by at least about30%, e.g., by at least about 40%, more preferably by at least about 50%,e.g., by at least about 75%, even more preferably by at least about100%, e.g., by at least about 150%, 200%, 250%, 300%, 400% or by atleast about 500%, compared to a reference situation without saidmodulation; or modulation may encompass a decrease or reduction in thevalue of the measured variable by at least about 10%, e.g., by at leastabout 20%, by at least about 30%, e.g., by at least about 40%, by atleast about 50%, e.g., by at least about 60%, by at least about 70%,e.g., by at least about 80%, by at least about 90%, e.g., by at leastabout 95%, such as by at least about 96%, 97%, 98%, 99% or even by 100%,compared to a reference situation without said modulation. Preferably,modulation may be specific or selective, hence, one or more desiredphenotypic aspects of an immune cell or immune cell population may bemodulated without substantially altering other (unintended, undesired)phenotypic aspect(s).

The term “immune cell” as used throughout this specification generallyencompasses any cell derived from a hematopoietic stem cell that plays arole in the immune response. The term is intended to encompass immunecells both of the innate or adaptive immune system. The immune cell asreferred to herein may be a leukocyte, at any stage of differentiation(e.g., a stem cell, a progenitor cell, a mature cell) or any activationstage. Immune cells include lymphocytes (such as natural killer cells,T-cells (including, e.g., thymocytes, Th or Tc; Th1, Th2, Th17, Thαβ,CD4⁺, CD8⁺, effector Th, memory Th, regulatory Th, CD4⁺/CD8⁺ thymocytes,CD4−/CD8− thymocytes, γδ T cells, etc.) or B-cells (including, e.g.,pro-B cells, early pro-B cells, late pro-B cells, pre-B cells, largepre-B cells, small pre-B cells, immature or mature B-cells, producingantibodies of any isotype, T1 B-cells, T2, B-cells, naïve B-cells, GCB-cells, plasmablasts, memory B-cells, plasma cells, follicular B-cells,marginal zone B-cells, B-1 cells, B-2 cells, regulatory B cells, etc.),such as for instance, monocytes (including, e.g., classical,non-classical, or intermediate monocytes), (segmented or banded)neutrophils, eosinophils, basophils, mast cells, histiocytes, microglia,including various subtypes, maturation, differentiation, or activationstages, such as for instance hematopoietic stem cells, myeloidprogenitors, lymphoid progenitors, myeloblasts, promyelocytes,myelocytes, metamyelocytes, monoblasts, promonocytes, lymphoblasts,prolymphocytes, small lymphocytes, macrophages (including, e.g., Kupffercells, stellate macrophages, M1 or M2 macrophages), (myeloid orlymphoid) dendritic cells (including, e.g., Langerhans cells,conventional or myeloid dendritic cells, plasmacytoid dendritic cells,mDC-1, mDC-2, Mo-DC, HP-DC, veiled cells), granulocytes,polymorphonuclear cells, antigen-presenting cells (APC), etc.

As used throughout this specification, “immune response” refers to aresponse by a cell of the immune system, such as a B cell, T cell (CD4⁺or CD8⁺), regulatory T cell, antigen-presenting cell, dendritic cell,monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, orneutrophil, to a stimulus. In some embodiments, the response is specificfor a particular antigen (an “antigen-specific response”), and refers toa response by a CD4 T cell, CD8 T cell, or B cell via theirantigen-specific receptor. In some embodiments, an immune response is aT cell response, such as a CD4⁺ response or a CD8⁺ response. Suchresponses by these cells can include, for example, cytotoxicity,proliferation, cytokine or chemokine production, trafficking, orphagocytosis, and can be dependent on the nature of the immune cellundergoing the response.

T cell response refers more specifically to an immune response in whichT cells directly or indirectly mediate or otherwise contribute to animmune response in a subject. T cell-mediated response may be associatedwith cell mediated effects, cytokine mediated effects, and even effectsassociated with B cells if the B cells are stimulated, for example, bycytokines secreted by T cells. By means of an example but withoutlimitation, effector functions of MEW class I restricted Cytotoxic Tlymphocytes (CTLs) may include cytokine and/or cytolytic capabilities,such as lysis of target cells presenting an antigen peptide recognizedby the T cell receptor (naturally-occurring TCR or geneticallyengineered TCR, e.g., chimeric antigen receptor, CAR), secretion ofcytokines, preferably IFN gamma, TNF alpha and/or or moreimmunostimulatory cytokines, such as IL-2, and/or antigenpeptide-induced secretion of cytotoxic effector molecules, such asgranzymes, perforins or granulysin. By means of example but withoutlimitation, for MEW class II restricted T helper (Th) cells, effectorfunctions may be antigen peptide-induced secretion of cytokines,preferably, IFN gamma, TNF alpha, IL-4, IL5, IL-10, and/or IL-2. Bymeans of example but without limitation, for T regulatory (Treg) cells,effector functions may be antigen peptide-induced secretion ofcytokines, preferably, IL-10, IL-35, and/or TGF-beta. B cell responserefers more specifically to an immune response in which B cells directlyor indirectly mediate or otherwise contribute to an immune response in asubject. Effector functions of B cells may include in particularproduction and secretion of antigen-specific antibodies by B cells(e.g., polyclonal B cell response to a plurality of the epitopes of anantigen (antigen-specific antibody response)), antigen presentation,and/or cytokine secretion.

During persistent immune activation, such as during uncontrolled tumorgrowth or chronic infections, subpopulations of immune cells,particularly of CD8+ or CD4+ T cells, become compromised to differentextents with respect to their cytokine and/or cytolytic capabilities.Such immune cells, particularly CD8+ or CD4+ T cells, are commonlyreferred to as “dysfunctional” or as “functionally exhausted” or“exhausted”. As used herein, the term “dysfunctional” or “functionalexhaustion” refer to a state of a cell where the cell does not performits usual function or activity in response to normal input signals, andincludes refractivity of immune cells to stimulation, such asstimulation via an activating receptor or a cytokine. Such a function oractivity includes, but is not limited to, proliferation (e.g., inresponse to a cytokine, such as IFN-gamma) or cell division, entranceinto the cell cycle, cytokine production, cytotoxicity, migration andtrafficking, phagocytotic activity, or any combination thereof. Normalinput signals can include, but are not limited to, stimulation via areceptor (e.g., T cell receptor, B cell receptor, co-stimulatoryreceptor). Unresponsive immune cells can have a reduction of at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% incytotoxic activity, cytokine production, proliferation, trafficking,phagocytotic activity, or any combination thereof, relative to acorresponding control immune cell of the same type. In some particularembodiments of the aspects described herein, a cell that isdysfunctional is a CD8+ T cell that expresses the CD8+ cell surfacemarker. Such CD8+ cells normally proliferate and produce cell killingenzymes, e.g., they can release the cytotoxins perforin, granzymes, andgranulysin. However, exhausted/dysfunctional T cells do not respondadequately to TCR stimulation, and display poor effector function,sustained expression of inhibitory receptors and a transcriptional statedistinct from that of functional effector or memory T cells.Dysfunction/exhaustion of T cells thus prevents optimal control ofinfection and tumors. Exhausted/dysfunctional immune cells, such as Tcells, such as CD8+ T cells, may produce reduced amounts of IFN-gamma,TNF-alpha and/or one or more immunostimulatory cytokines, such as IL-2,compared to functional immune cells. Exhausted/dysfunctional immunecells, such as T cells, such as CD8+ T cells, may further produce(increased amounts of) one or more immunosuppressive transcriptionfactors or cytokines, such as IL-10 and/or Foxp3, compared to functionalimmune cells, thereby contributing to local immunosuppression.Dysfunctional CD8+ T cells can be both protective and detrimentalagainst disease control. As used herein, a “dysfunctional immune state”refers to an overall suppressive immune state in a subject ormicroenvironment of the subject (e.g., tumor microenvironment). Forexample, increased IL-10 production leads to suppression of other immunecells in a population of immune cells.

CD8+ T cell function is associated with their cytokine profiles. It hasbeen reported that effector CD8+ T cells with the ability tosimultaneously produce multiple cytokines (polyfunctional CD8+ T cells)are associated with protective immunity in patients with controlledchronic viral infections as well as cancer patients responsive to immunetherapy (Spranger et al., 2014, J. Immunother. Cancer, vol. 2, 3). Inthe presence of persistent antigen CD8+ T cells were found to have lostcytolytic activity completely over time (Moskophidis et al., 1993,Nature, vol. 362, 758-761). It was subsequently found that dysfunctionalT cells can differentially produce IL-2, TNFa and IFNg in a hierarchicalorder (Wherry et al., 2003, J. Virol., vol. 77, 4911-4927). Decoupleddysfunctional and activated CD8+ cell states have also been described(see, e.g., Singer, et al. (2016). A Distinct Gene Module forDysfunction Uncoupled from Activation in Tumor-Infiltrating T Cells.Cell 166, 1500-1511 e1509; WO/2017/075478; and WO/2018/049025).

The invention provides compositions and methods for modulating T cellbalance. The invention provides T cell modulating agents that modulate Tcell balance. For example, in some embodiments, the invention provides Tcell modulating agents and methods of using these T cell modulatingagents to regulate, influence or otherwise impact the level of and/orbalance between T cell types, e.g., between Th17 and other T cell types,for example, Th1-like cells. For example, in some embodiments, theinvention provides T cell modulating agents and methods of using these Tcell modulating agents to regulate, influence or otherwise impact thelevel of and/or balance between Th17 activity and inflammatorypotential. As used herein, terms such as “Th17 cell” and/or “Th17phenotype” and all grammatical variations thereof refer to adifferentiated T helper cell that expresses one or more cytokinesselected from the group the consisting of interleukin 17A (IL-17A),interleukin 17F (IL-17F), and interleukin 17A/F heterodimer (IL17-AF).As used herein, terms such as “TH1 cell” and/or “TH1 phenotype” and allgrammatical variations thereof refer to a differentiated T helper cellthat expresses interferon gamma (IFNγ). As used herein, terms such as“Th2 cell” and/or “Th2 phenotype” and all grammatical variations thereofrefer to a differentiated T helper cell that expresses one or morecytokines selected from the group the consisting of interleukin 4(IL-4), interleukin 5 (IL-5) and interleukin 13 (IL-13). As used herein,terms such as “Treg cell” and/or “Treg phenotype” and all grammaticalvariations thereof refer to a differentiated T cell that expressesFoxp3.

Depending on the cytokines used for differentiation, in vitro polarizedTh17 cells can either cause severe autoimmune responses upon adoptivetransfer (pathogenic Th17 cells') or have little or no effect ininducing autoimmune disease (non-pathogenic cells') (Ghoreschi et al.,2010; and Lee et al., 2012 “Induction and molecular signature ofpathogenic Th17 cells,” Nature Immunology, vol. 13(10): 991-999). Invitro differentiation of naïve CD4 T cells in the presence ofTGF-β1+IL-6 induces an IL-17A and IL-10 producing population of Th17cells, that are generally nonpathogenic, whereas activation of naïve Tcells in the presence of IL-1β+IL-6+IL-23 or TGF-β3+IL-6 induces a Tcell population that produces IL-17A and IFN-γ, and are potent inducersof autoimmune disease induction (Ghoreschi et al., 2010, Lee et al.,2012).

As used herein, terms such as “pathogenic Th17 cell” and/or “pathogenicTh17 phenotype” and all grammatical variations thereof refer to Th17cells that, exhibit a distinct pathogenic signature where one or more ofthe following genes or products of these genes is upregulated in Th17cells: Cxcl3, Il22, Il3, Ccl4, Gzmb, Lrmp, Ccl5, Casp1, Csf2, Ccl3,Tbx21, Icos, Il7r, Stat4, Lgals3 or Lag3. In certain embodiments, thepathogenic signature is elevated in TGF-β3-induced Th17 cells ascompared to TGF-β1-induced Th17 cells. As used herein, terms such as“non-pathogenic Th17 cell” and/or “non-pathogenic Th17 phenotype” andall grammatical variations thereof refer to Th17 cells that exhibit adistinct non-pathogenic signature where one or more of the followinggenes or products of these genes is up-regulated in Th17 cells: Il6st,Il1rn, lkzf3, Maf, Ahr, Il9 or Il10. In certain embodiments, thenon-pathogenic signature is elevated in TGF-β1-induced Th17 cells ascompared to TGF-β33-induced Th17 cells. In certain embodiments, wheninduced in the presence of TGF-β3, Th17 cells express a decreased levelof one or more genes selected from IL6st, IL1rn, Ikzf3, Maf, Ahr, IL9and IL10, as compared to the level of expression in TGF-β3-induced Th17cells.

A dynamic regulatory network controls Th17 differentiation (See e.g.,Yosef et al., Dynamic regulatory network controlling Th17 celldifferentiation, Nature, vol. 496: 461-468 (2013); Wang et al., CD5L/AIMRegulates Lipid Biosynthesis and Restrains Th17 Cell Pathogenicity, CellVolume 163, Issue 6, p1413-142′7, 3 Dec. 2015; Gaublomme et al.,Single-Cell Genomics Unveils Critical Regulators of Th17 CellPathogenicity, Cell Volume 163, Issue 6, p1400-1412, 3 Dec. 2015; andInternational Patent Publication Nos. WO2016138488A2, WO2015130968,WO/2012/048265, WO/2014/145631 and WO/2014/134351, the contents of whichare hereby incorporated by reference in their entirety).

Methods of Determining Tumor Resistance to an Immune Response

In certain embodiments, detection of interacting cell types indicatesresistance to an immune response. In certain embodiments, detection ofinteracting cells in the TME may indicate that the subject may beresponsive to an immunotherapy. In one example embodiment, expression ofreceptors or ligands on tumor cells that can interact with immune cellsin the TME indicates that the tumor can modulate the TME to suppress ananti-tumor immune response. In certain embodiments, a tumor sample, suchas a tissue section can be obtained and stained for any of VEGFB, TFPI,MFGE8, ADAM12 and FBLN1.

Diagnostic, Prognostic and Therapeutic Methods

The invention provides biomarkers for the identification, diagnosis,prognosis and manipulation of disease phenotypes, for use in a varietyof diagnostic and/or therapeutic indications. Biomarkers in the contextof the present invention encompasses, without limitation nucleic acids,proteins, reaction products, and metabolites, together with theirpolymorphisms, mutations, variants, modifications, subunits, fragments,and other analytes or sample-derived measures. In certain embodiments,biomarkers include the signature genes or signature gene products,and/or cells as described herein.

Therapeutic Agents

In certain embodiments, one or more of the biomarkers is a therapeutictarget. In certain embodiments, the present invention provides for oneor more therapeutic agents targeting one or more therapeutic targets totreat cancer. Targeting the identified cell interactions may provide forenhanced or otherwise previously unknown activity in the treatment ofdisease. In certain embodiments, the agents are used to modulate celltypes (e.g., shifting signatures). In certain embodiments, the one ormore therapeutic agents target genes differentially expressed in thecell types over time or tumor growth. In certain embodiments, the one ormore agents comprises a small molecule inhibitor, small moleculedegrader (e.g., ATTEC, AUTAC, LYTAC, or PROTAC), genetic modifyingagent, antibody, antibody fragment, antibody-like protein scaffold,aptamer, protein, or any combination thereof.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested. As used herein “treating”includes ameliorating, curing, preventing it from becoming worse,slowing the rate of progression, or preventing the disorder fromre-occurring (i.e., to prevent a relapse).

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

For example, in methods for treating cancer in a subject, an effectiveamount of a combination of agents is any amount that provides ananti-tumor effect, such as reduces or prevents proliferation of a cancercell or makes a cancer cell responsive to an immunotherapy.

Standard of Care

Aspects of the invention involve modifying the therapy within a standardof care based on the detection of any of the biomarkers as describedherein. In one embodiment, therapy comprising an agent is administeredwithin a standard of care where addition of the agent is synergisticwithin the steps of the standard of care. In one embodiment, the agenttargets and/or shifts a tumor to an immunotherapy responder phenotype.In one embodiment, the agent inhibits expression or activity of one ormore transcription factors capable of regulating a gene program. In oneembodiment, the agent targets tumor cells expressing a gene program. Theterm “standard of care” as used herein refers to the current treatmentthat is accepted by medical experts as a proper treatment for a certaintype of disease and that is widely used by healthcare professionals.Standard of care is also called best practice, standard medical care,and standard therapy. Standards of care for cancer generally includesurgery, lymph node removal, radiation, chemotherapy, targetedtherapies, antibodies targeting the tumor, and immunotherapy.Immunotherapy can include checkpoint blockers (CBP), chimeric antigenreceptors (CARs), and adoptive T-cell therapy. The standards of care forthe most common cancers can be found on the website of National CancerInstitute (www.cancer.gov/cancertopics). A treatment clinical trial is aresearch study meant to help improve current treatments or obtaininformation on new treatments for patients with cancer. When clinicaltrials show that a new treatment is better than the standard treatment,the new treatment may be considered the new standard treatment.

The term “Adjuvant therapy” as used herein refers to any treatment givenafter primary therapy to increase the chance of long-term disease-freesurvival. The term “Neoadjuvant therapy” as used herein refers to anytreatment given before primary therapy. The term “Primary therapy” asused herein refers to the main treatment used to reduce or eliminate thecancer. In certain embodiments, an agent that shifts a tumor to aresponder phenotype are provided as a neoadjuvant before CPB therapy.

Checkpoint Blockade Therapy

In certain embodiments, the one or more agents described herein areadministered to enhance the effectiveness of an immunotherapy, e.g., bypreventing suppression of an immune response. Immunotherapy can includecheckpoint blockers (CBP), chimeric antigen receptors (CARs), andadoptive T-cell therapy. Antibodies that block the activity ofcheckpoint receptors, including CTLA-4, PD-1, Tim-3, Lag-3, and TIGIT,either alone or in combination, have been associated with improvedeffector CD8⁺ T cell responses in multiple pre-clinical cancer models(Johnston et al., 2014. The immunoreceptor TIGIT regulates antitumor andantiviral CD8(+) T cell effector function. Cancer cell 26, 923-937;Ngiow et al., 2011. Anti-TIM3 antibody promotes T cellIFN-gamma-mediated antitumor immunity and suppresses established tumors.Cancer research 71, 3540-3551; Sakuishi et al., 2010. Targeting Tim-3and PD-1 pathways to reverse T cell exhaustion and restore anti-tumorimmunity. The Journal of experimental medicine 207, 2187-2194; and Wooet al., 2012. Immune inhibitory molecules LAG-3 and PD-1 synergisticallyregulate T-cell function to promote tumoral immune escape. Cancerresearch 72, 917-927). Similarly, blockade of CTLA-4 and PD-1 inpatients (Brahmer et al., 2012. Safety and activity of anti-PD-Llantibody in patients with advanced cancer. The New England journal ofmedicine 366, 2455-2465; Hodi et al., 2010. Improved survival withipilimumab in patients with metastatic melanoma. The New England journalof medicine 363, 711-723; Schadendorf et al., 2015. Pooled Analysis ofLong-Term Survival Data From Phase II and Phase III Trials of Ipilimumabin Unresectable or Metastatic Melanoma. Journal of clinical oncology:official journal of the American Society of Clinical Oncology 33,1889-1894; Topalian et al., 2012. Safety, activity, and immunecorrelates of anti-PD-1 antibody in cancer. The New England journal ofmedicine 366, 2443-2454; and Wolchok et al., 2017. Overall Survival withCombined Nivolumab and Ipilimumab in Advanced Melanoma. The New Englandjournal of medicine 377, 1345-1356) has shown increased frequencies ofproliferating T cells, often with specificity for tumor antigens, aswell as increased CD8⁺ T cell effector function (Ayers et al., 2017.IFN-gamma-related mRNA profile predicts clinical response to PD-1blockade. The Journal of clinical investigation 127, 2930-2940; Das etal., 2015. Combination therapy with anti-CTLA-4 and anti-PD-1 leads todistinct immunologic changes in vivo. Journal of immunology 194,950-959; Gubin et al., 2014. Checkpoint blockade cancer immunotherapytargets tumour-specific mutant antigens. Nature 515, 577-581; Huang etal., 2017. T-cell invigoration to tumour burden ratio associated withanti-PD-1 response. Nature 545, 60-65; Kamphorst et al., 2017.Proliferation of PD-1+CD8 T cells in peripheral blood afterPD-1-targeted therapy in lung cancer patients. Proceedings of theNational Academy of Sciences of the United States of America 114,4993-4998; Kvistborg et al., 2014. Anti-CTLA-4 therapy broadens themelanoma-reactive CD8+ T cell response. Science translational medicine6, 254ra128; van Rooij et al., 2013. Tumor exome analysis revealsneoantigen-specific T-cell reactivity in an ipilimumab-responsivemelanoma. Journal of clinical oncology: official journal of the AmericanSociety of Clinical Oncology 31, e439-442; and Yuan et al., 2008. CTLA-4blockade enhances polyfunctional NY-ESO-1 specific T cell responses inmetastatic melanoma patients with clinical benefit. Proceedings of theNational Academy of Sciences of the United States of America 105,20410-20415). Accordingly, the success of checkpoint receptor blockadehas been attributed to the binding of blocking antibodies to checkpointreceptors expressed on dysfunctional CD8+ T cells and restoring effectorfunction in these cells. The check point blockade therapy may be aninhibitor of any check point protein described herein. The checkpointblockade therapy may comprise anti-TIM3, anti-CTLA4, anti-PD-L1,anti-PD1, anti-TIGIT, anti-LAG3, or combinations thereof. Anti-PD1antibodies are disclosed in U.S. Pat. No. 8,735,553. Antibodies to LAG-3are disclosed in U.S. Pat. No. 9,132,281. Anti-CTLA4 antibodies aredisclosed in U.S. Pat. No. 9,327,014; U.S. Pat. No. 9,320,811; and U.S.Pat. No. 9,062,111. Specific check point inhibitors include, but are notlimited to anti-CTLA4 antibodies (e.g., Ipilimumab and tremelimumab),anti-PD-1 antibodies (e.g., Nivolumab, Pembrolizumab), and anti-PD-L1antibodies (e.g., Atezolizumab).

Antibodies

In certain embodiments, the one or more agents is an antibody. The term“antibody” is used interchangeably with the term “immunoglobulin”herein, and includes intact antibodies, fragments of antibodies, e.g.,Fab, F(ab′)2 fragments, and intact antibodies and fragments that havebeen mutated either in their constant and/or variable region (e.g.,mutations to produce chimeric, partially humanized, or fully humanizedantibodies, as well as to produce antibodies with a desired trait, e.g.,enhanced binding and/or reduced FcR binding). The term “fragment” refersto a part or portion of an antibody or antibody chain comprising feweramino acid residues than an intact or complete antibody or antibodychain. Fragments can be obtained via chemical or enzymatic treatment ofan intact or complete antibody or antibody chain. Fragments can also beobtained by recombinant means. Exemplary fragments include Fab, Fab′,F(ab′)2, Fabc, Fd, dAb, V_(HH) and scFv and/or Fv fragments.

As used herein, a preparation of antibody protein having less than about50% of non-antibody protein (also referred to herein as a “contaminatingprotein”), or of chemical precursors, is considered to be “substantiallyfree.” 40%, 30%, 20%, 10% and more preferably 5% (by dry weight), ofnon-antibody protein, or of chemical precursors is considered to besubstantially free. When the antibody protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 30%, preferably less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume or mass of the protein preparation.

The term “antigen-binding fragment” refers to a polypeptide fragment ofan immunoglobulin or antibody that binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding). As such these antibodiesor fragments thereof are included in the scope of the invention,provided that the antibody or fragment binds specifically to a targetmolecule.

It is intended that the term “antibody” encompass any Ig class or any Igsubclass (e.g. the IgG1, IgG2, IgG3, and IgG4 subclasses of IgG)obtained from any source (e.g., humans and non-human primates, and inrodents, lagomorphs, caprines, bovines, equines, ovines, etc.).

The term “Ig class” or “immunoglobulin class”, as used herein, refers tothe five classes of immunoglobulin that have been identified in humansand higher mammals, IgG, IgM, IgA, IgD, and IgE. The term “Ig subclass”refers to the two subclasses of IgM (H and L), three subclasses of IgA(IgA1, IgA2, and secretory IgA), and four subclasses of IgG (IgG1, IgG2,IgG3, and IgG4) that have been identified in humans and higher mammals.The antibodies can exist in monomeric or polymeric form; for example,1gM antibodies exist in pentameric form, and IgA antibodies exist inmonomeric, dimeric or multimeric form.

The term “IgG subclass” refers to the four subclasses of immunoglobulinclass IgG-IgG1, IgG2, IgG3, and IgG4 that have been identified in humansand higher mammals by the heavy chains of the immunoglobulins, V1-γ4,respectively. The term “single-chain immunoglobulin” or “single-chainantibody” (used interchangeably herein) refers to a protein having atwo-polypeptide chain structure consisting of a heavy and a light chain,said chains being stabilized, for example, by interchain peptidelinkers, which has the ability to specifically bind antigen. The term“domain” refers to a globular region of a heavy or light chainpolypeptide comprising peptide loops (e.g., comprising 3 to 4 peptideloops) stabilized, for example, by β pleated sheet and/or intrachaindisulfide bond. Domains are further referred to herein as “constant” or“variable”, based on the relative lack of sequence variation within thedomains of various class members in the case of a “constant” domain, orthe significant variation within the domains of various class members inthe case of a “variable” domain. Antibody or polypeptide “domains” areoften referred to interchangeably in the art as antibody or polypeptide“regions”. The “constant” domains of an antibody light chain arereferred to interchangeably as “light chain constant regions”, “lightchain constant domains”, “CL” regions or “CL” domains. The “constant”domains of an antibody heavy chain are referred to interchangeably as“heavy chain constant regions”, “heavy chain constant domains”, “CH”regions or “CH” domains). The “variable” domains of an antibody lightchain are referred to interchangeably as “light chain variable regions”,“light chain variable domains”, “VL” regions or “VL” domains). The“variable” domains of an antibody heavy chain are referred tointerchangeably as “heavy chain constant regions”, “heavy chain constantdomains”, “VH” regions or “VH” domains).

The term “region” can also refer to a part or portion of an antibodychain or antibody chain domain (e.g., a part or portion of a heavy orlight chain or a part or portion of a constant or variable domain, asdefined herein), as well as more discrete parts or portions of saidchains or domains. For example, light and heavy chains or light andheavy chain variable domains include “complementarity determiningregions” or “CDRs” interspersed among “framework regions” or “FRs”, asdefined herein.

The term “conformation” refers to the tertiary structure of a protein orpolypeptide (e.g., an antibody, antibody chain, domain or regionthereof). For example, the phrase “light (or heavy) chain conformation”refers to the tertiary structure of a light (or heavy) chain variableregion, and the phrase “antibody conformation” or “antibody fragmentconformation” refers to the tertiary structure of an antibody orfragment thereof.

The term “antibody-like protein scaffolds” or “engineered proteinscaffolds” broadly encompasses proteinaceous non-immunoglobulinspecific-binding agents, typically obtained by combinatorial engineering(such as site-directed random mutagenesis in combination with phagedisplay or other molecular selection techniques). Usually, suchscaffolds are derived from robust and small soluble monomeric proteins(such as Kunitz inhibitors or lipocalins) or from a stably foldedextra-membrane domain of a cell surface receptor (such as protein A,fibronectin or the ankyrin repeat).

Such scaffolds have been extensively reviewed in Binz et al.(Engineering novel binding proteins from nonimmunoglobulin domains. NatBiotechnol 2005, 23:1257-1268), Gebauer and Skerra (Engineered proteinscaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol.2009, 13:245-55), Gill and Damle (Biopharmaceutical drug discovery usingnovel protein scaffolds. Curr Opin Biotechnol 2006, 17:653-658), Skerra(Engineered protein scaffolds for molecular recognition. J Mol Recognit2000, 13:167-187), and Skerra (Alternative non-antibody scaffolds formolecular recognition. Curr Opin Biotechnol 2007, 18:295-304), andinclude without limitation affibodies, based on the Z-domain ofstaphylococcal protein A, a three-helix bundle of 58 residues providingan interface on two of its alpha-helices (Nygren, Alternative bindingproteins: Affibody binding proteins developed from a small three-helixbundle scaffold. FEBS J 2008, 275:2668-2676); engineered Kunitz domainsbased on a small (ca. 58 residues) and robust, disulphide-crosslinkedserine protease inhibitor, typically of human origin (e.g. LACI-D1),which can be engineered for different protease specificities (Nixon andWood, Engineered protein inhibitors of proteases. Curr Opin Drug DiscovDev 2006, 9:261-268); monobodies or adnectins based on the 10thextracellular domain of human fibronectin III (10Fn3), which adopts anIg-like beta-sandwich fold (94 residues) with 2-3 exposed loops, butlacks the central disulphide bridge (Koide and Koide, Monobodies:antibody mimics based on the scaffold of the fibronectin type IIIdomain. Methods Mol Biol 2007, 352:95-109); anticalins derived from thelipocalins, a diverse family of eight-stranded beta-barrel proteins (ca.180 residues) that naturally form binding sites for small ligands bymeans of four structurally variable loops at the open end, which areabundant in humans, insects, and many other organisms (Skerra,Alternative binding proteins: Anticalins—harnessing the structuralplasticity of the lipocalin ligand pocket to engineer novel bindingactivities. FEBS J 2008, 275:2677-2683); DARPins, designed ankyrinrepeat domains (166 residues), which provide a rigid interface arisingfrom typically three repeated beta-turns (Stumpp et al., DARPins: a newgeneration of protein therapeutics. Drug Discov Today 2008, 13:695-701);avimers (multimerized LDLR-A module) (Silverman et al., Multivalentavimer proteins evolved by exon shuffling of a family of human receptordomains. Nat Biotechnol 2005, 23:1556-1561); and cysteine-rich knottinpeptides (Kolmar, Alternative binding proteins: biological activity andtherapeutic potential of cystine-knot miniproteins. FEBS J 2008,275:2684-2690).

“Specific binding” of an antibody means that the antibody exhibitsappreciable affinity for a particular antigen or epitope and, generally,does not exhibit significant cross reactivity. “Appreciable” bindingincludes binding with an affinity of at least 25 μM. Antibodies withaffinities greater than 1×10⁷ M⁻¹ (or a dissociation coefficient of 1 μMor less or a dissociation coefficient of lnm or less) typically bindwith correspondingly greater specificity. Values intermediate of thoseset forth herein are also intended to be within the scope of the presentinvention and antibodies of the invention bind with a range ofaffinities, for example, 100 nM or less, 75 nM or less, 50 nM or less,25 nM or less, for example 10 nM or less, 5 nM or less, 1 nM or less, orin embodiments 500 pM or less, 100 pM or less, 50 pM or less or 25 pM orless. An antibody that “does not exhibit significant crossreactivity” isone that will not appreciably bind to an entity other than its target(e.g., a different epitope or a different molecule). For example, anantibody that specifically binds to a target molecule will appreciablybind the target molecule but will not significantly react withnon-target molecules or peptides. An antibody specific for a particularepitope will, for example, not significantly crossreact with remoteepitopes on the same protein or peptide. Specific binding can bedetermined according to any art-recognized means for determining suchbinding. Preferably, specific binding is determined according toScatchard analysis and/or competitive binding assays.

As used herein, the term “affinity” refers to the strength of thebinding of a single antigen-combining site with an antigenicdeterminant. Affinity depends on the closeness of stereochemical fitbetween antibody combining sites and antigen determinants, on the sizeof the area of contact between them, on the distribution of charged andhydrophobic groups, etc. Antibody affinity can be measured byequilibrium dialysis or by the kinetic BIACORE™ method. The dissociationconstant, Kd, and the association constant, Ka, are quantitativemeasures of affinity.

As used herein, the term “monoclonal antibody” refers to an antibodyderived from a clonal population of antibody-producing cells (e.g., Blymphocytes or B cells) which is homogeneous in structure and antigenspecificity. The term “polyclonal antibody” refers to a plurality ofantibodies originating from different clonal populations ofantibody-producing cells which are heterogeneous in their structure andepitope specificity but which recognize a common antigen. Monoclonal andpolyclonal antibodies may exist within bodily fluids, as crudepreparations, or may be purified, as described herein.

The term “binding portion” of an antibody (or “antibody portion”)includes one or more complete domains, e.g., a pair of complete domains,as well as fragments of an antibody that retain the ability tospecifically bind to a target molecule. It has been shown that thebinding function of an antibody can be performed by fragments of afull-length antibody. Binding fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intactimmunoglobulins. Binding fragments include Fab, Fab′, F(ab′)2, Fabc, Fd,dAb, Fv, single chains, single-chain antibodies, e.g., scFv, and singledomain antibodies.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, FR residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

Examples of portions of antibodies or epitope-binding proteinsencompassed by the present definition include: (i) the Fab fragment,having V_(L), C_(L), V_(H) and C_(H)1 domains; (ii) the Fab′ fragment,which is a Fab fragment having one or more cysteine residues at theC-terminus of the C_(H)1 domain; (iii) the Fd fragment having V_(H) andC_(H)1 domains; (iv) the Fd′ fragment having VH and C_(H)1 domains andone or more cysteine residues at the C-terminus of the CHI domain; (v)the Fv fragment having the V_(L) and V_(H) domains of a single arm of anantibody; (vi) the dAb fragment (Ward et al., 341 Nature 544 (1989))which consists of a V_(H) domain or a V_(L) domain that binds antigen;(vii) isolated CDR regions or isolated CDR regions presented in afunctional framework; (viii) F(ab′)₂ fragments which are bivalentfragments including two Fab′ fragments linked by a disulphide bridge atthe hinge region; (ix) single chain antibody molecules (e.g., singlechain Fv; scFv) (Bird et al., 242 Science 423 (1988); and Huston et al.,85 PNAS 5879 (1988)); (x) “diabodies” with two antigen binding sites,comprising a heavy chain variable domain (V_(H)) connected to a lightchain variable domain (V_(L)) in the same polypeptide chain (see, e.g.,EP 404,097; WO 93/11161; Hollinger et al., 90 PNAS 6444 (1993)); (xi)“linear antibodies” comprising a pair of tandem Fd segments(V_(H)-C_(h)1-VH-C_(h)1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.,Protein Eng. 8(10):1057-62 (1995); and U.S. Pat. No. 5,641,870).

As used herein, a “blocking” antibody or an antibody “antagonist” is onewhich inhibits or reduces biological activity of the antigen(s) itbinds. In certain embodiments, the blocking antibodies or antagonistantibodies or portions thereof described herein completely inhibit thebiological activity of the antigen(s).

Antibodies may act as agonists or antagonists of the recognizedpolypeptides. For example, the present invention includes antibodieswhich disrupt receptor/ligand interactions either partially or fully.The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orof one of its down-stream substrates by immunoprecipitation followed bywestern blot analysis. In specific embodiments, antibodies are providedthat inhibit ligand activity or receptor activity by at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex. Likewise, encompassed by theinvention are neutralizing antibodies which bind the ligand and preventbinding of the ligand to the receptor, as well as antibodies which bindthe ligand, thereby preventing receptor activation, but do not preventthe ligand from binding the receptor. Further included in the inventionare antibodies which activate the receptor. These antibodies may act asreceptor agonists, i.e., potentiate or activate either all or a subsetof the biological activities of the ligand-mediated receptor activation,for example, by inducing dimerization of the receptor. The antibodiesmay be specified as agonists, antagonists or inverse agonists forbiological activities comprising the specific biological activities ofthe peptides disclosed herein. The antibody agonists and antagonists canbe made using methods known in the art. See, e.g., PCT publication WO96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988(1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al.,J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res.58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179(1998); Prat et al., J. Cell. Sci. III (Pt2):237-247 (1998); Pitard etal., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al.,Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem.272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995);Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(1):14-20 (1996).

The antibodies as defined for the present invention include derivativesthat are modified, i.e., by the covalent attachment of any type ofmolecule to the antibody such that covalent attachment does not preventthe antibody from generating an anti-idiotypic response. For example,but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited tospecific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

Simple binding assays can be used to screen for or detect agents thatbind to a target protein, or disrupt the interaction between proteins(e.g., a receptor and a ligand). Because certain targets of the presentinvention are transmembrane proteins, assays that use the soluble formsof these proteins rather than full-length protein can be used, in someembodiments. Soluble forms include, for example, those lacking thetransmembrane domain and/or those comprising the IgV domain or fragmentsthereof which retain their ability to bind their cognate bindingpartners. Further, agents that inhibit or enhance protein interactionsfor use in the compositions and methods described herein, can includerecombinant peptido-mimetics.

Detection methods useful in screening assays include antibody-basedmethods, detection of a reporter moiety, detection of cytokines asdescribed herein, and detection of a gene signature as described herein.

Another variation of assays to determine binding of a receptor proteinto a ligand protein is through the use of affinity biosensor methods.Such methods may be based on the piezoelectric effect, electrochemistry,or optical methods, such as ellipsometry, optical wave guidance, andsurface plasmon resonance (SPR).

Bi-Specific Antibodies

In certain embodiments, the one or more therapeutic agents can bebi-specific antigen-binding constructs, e.g., bi-specific antibodies(bsAb) or BiTEs, that bind two antigens (see, e.g., Suurs et al., Areview of bispecific antibodies and antibody constructs in oncology andclinical challenges. Pharmacol Ther. 2019 September; 201:103-119; andHuehls, et al., Bispecific T cell engagers for cancer immunotherapy.Immunol Cell Biol. 2015 March; 93(3): 290-296). The bi-specificantigen-binding construct includes two antigen-binding polypeptideconstructs, e.g., antigen binding domains, wherein at least onepolypeptide construct specifically binds to a surface protein. In someembodiments, the antigen-binding construct is derived from knownantibodies or antigen-binding constructs. In some embodiments, theantigen-binding polypeptide constructs comprise two antigen bindingdomains that comprise antibody fragments. In some embodiments, the firstantigen binding domain and second antigen binding domain eachindependently comprises an antibody fragment selected from the group of:an scFv, a Fab, and an Fc domain. The antibody fragments may be the sameformat or different formats from each other. For example, in someembodiments, the antigen-binding polypeptide constructs comprise a firstantigen binding domain comprising an scFv and a second antigen bindingdomain comprising a Fab. In some embodiments, the antigen-bindingpolypeptide constructs comprise a first antigen binding domain and asecond antigen binding domain, wherein both antigen binding domainscomprise an scFv. In some embodiments, the first and second antigenbinding domains each comprise a Fab. In some embodiments, the first andsecond antigen binding domains each comprise an Fc domain. Anycombination of antibody formats is suitable for the bi-specific antibodyconstructs disclosed herein.

In certain embodiments, immune cells can be engaged to other immunecells. In certain embodiments, immune cells are targeted with a bsAbhaving affinity for both the immune cell and a payload (e.g.,therapeutic agent). In certain embodiments, two targets are disrupted onan immune cell by the bsAb. By means of an example, an agent, such as abi-specific antibody, specifically binds to a gene product expressed onthe cell surface of the immune cells. In certain embodiments, immunecells can be targeted.

Aptamers

In certain embodiments, the one or more agents is an aptamer. Nucleicacid aptamers are nucleic acid species that have been engineered throughrepeated rounds of in vitro selection or equivalently, SELEX (systematicevolution of ligands by exponential enrichment) to bind to variousmolecular targets such as small molecules, proteins, nucleic acids,cells, tissues and organisms. Nucleic acid aptamers have specificbinding affinity to molecules through interactions other than classicWatson-Crick base pairing. Aptamers are useful in biotechnological andtherapeutic applications as they offer molecular recognition propertiessimilar to antibodies. In addition to their discriminate recognition,aptamers offer advantages over antibodies as they can be engineeredcompletely in a test tube, are readily produced by chemical synthesis,possess desirable storage properties, and elicit little or noimmunogenicity in therapeutic applications. In certain embodiments, RNAaptamers may be expressed from a DNA construct. In other embodiments, anucleic acid aptamer may be linked to another polynucleotide sequence.The polynucleotide sequence may be a double stranded DNA polynucleotidesequence. The aptamer may be covalently linked to one strand of thepolynucleotide sequence. The aptamer may be ligated to thepolynucleotide sequence. The polynucleotide sequence may be configured,such that the polynucleotide sequence may be linked to a solid supportor ligated to another polynucleotide sequence.

Aptamers, like peptides generated by phage display or monoclonalantibodies (“mAbs”), are capable of specifically binding to selectedtargets and modulating the target's activity, e.g., through binding,aptamers may block their target's ability to function. A typical aptameris 10-15 kDa in size (30-45 nucleotides), binds its target withsub-nanomolar affinity, and discriminates against closely relatedtargets (e.g., aptamers will typically not bind other proteins from thesame gene family). Structural studies have shown that aptamers arecapable of using the same types of binding interactions (e.g., hydrogenbonding, electrostatic complementarity, hydrophobic contacts, stericexclusion) that drives affinity and specificity in antibody-antigencomplexes.

Aptamers have a number of desirable characteristics for use in researchand as therapeutics and diagnostics including high specificity andaffinity, biological efficacy, and excellent pharmacokinetic properties.In addition, they offer specific competitive advantages over antibodiesand other protein biologics. Aptamers are chemically synthesized and arereadily scaled as needed to meet production demand for research,diagnostic or therapeutic applications. Aptamers are chemically robust.They are intrinsically adapted to regain activity following exposure tofactors such as heat and denaturants and can be stored for extendedperiods (>1 yr) at room temperature as lyophilized powders. Not beingbound by a theory, aptamers bound to a solid support or beads may bestored for extended periods.

Oligonucleotides in their phosphodiester form may be quickly degraded byintracellular and extracellular enzymes such as endonucleases andexonucleases. Aptamers can include modified nucleotides conferringimproved characteristics on the ligand, such as improved in vivostability or improved delivery characteristics. Examples of suchmodifications include chemical substitutions at the ribose and/orphosphate and/or base positions. SELEX identified nucleic acid ligandscontaining modified nucleotides are described, e.g., in U.S. Pat. No.5,660,985, which describes oligonucleotides containing nucleotidederivatives chemically modified at the 2′ position of ribose, 5 positionof pyrimidines, and 8 position of purines, U.S. Pat. No. 5,756,703 whichdescribes oligonucleotides containing various 2′ -modified pyrimidines,and U.S. Pat. No. 5,580,737 which describes highly specific nucleic acidligands containing one or more nucleotides modified with 2′-amino(2′-NH₂), 2′-fluoro (2′-F), and/or 2′-0-methyl (2′-OMe) substituents.Modifications of aptamers may also include, modifications at exocyclicamines, substitution of 4-thiouridine, substitution of 5-bromo or5-iodo-uracil; backbone modifications, phosphorothioate or allylphosphate modifications, methylations, and unusual base-pairingcombinations such as the isobases isocytidine and isoguanosine.Modifications can also include 3′ and 5′ modifications such as capping.As used herein, the term phosphorothioate encompasses one or morenon-bridging oxygen atoms in a phosphodiester bond replaced by one ormore sulfur atoms. In further embodiments, the oligonucleotides comprisemodified sugar groups, for example, one or more of the hydroxyl groupsis replaced with halogen, aliphatic groups, or functionalized as ethersor amines. In one embodiment, the 2′-position of the furanose residue issubstituted by any of an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl,or halo group. Methods of synthesis of 2′-modified sugars are described,e.g., in Sproat, et al., Nucl. Acid Res. 19:733-738 (1991); Cotten, etal, Nucl. Acid Res. 19:2629-2635 (1991); and Hobbs, et al, Biochemistry12:5138-5145 (1973). Other modifications are known to one of ordinaryskill in the art. In certain embodiments, aptamers include aptamers withimproved off-rates as described in International Patent Publication No.WO 2009012418, “Method for generating aptamers with improved off-rates,”incorporated herein by reference in its entirety. In certain embodimentsaptamers are chosen from a library of aptamers. Such libraries include,but are not limited to those described in Rohloff et al., “Nucleic AcidLigands With Protein-like Side Chains: Modified Aptamers and Their Useas Diagnostic and Therapeutic Agents,” Molecular Therapy Nucleic Acids(2014) 3, e201. Aptamers are also commercially available (see, e.g.,SomaLogic, Inc., Boulder, Colorado). In certain embodiments, the presentinvention may utilize any aptamer containing any modification asdescribed herein.

Small Molecules

In certain embodiments, the one or more agents is a small molecule. Theterm “small molecule” refers to compounds, preferably organic compounds,with a size comparable to those organic molecules generally used inpharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, peptides, nucleic acids, etc.). Preferred small organicmolecules range in size up to about 5000 Da, e.g., up to about 4000,preferably up to 3000 Da, more preferably up to 2000 Da, even morepreferably up to about 1000 Da, e.g., up to about 900, 800, 700, 600 orup to about 500 Da. In certain embodiments, the small molecule may actas an antagonist or agonist (e.g., blocking an enzyme active site oractivating a receptor by binding to a ligand binding site).

One type of small molecule applicable to the present invention is adegrader molecule (see, e.g., Ding, et al., Emerging New Concepts ofDegrader Technologies, Trends Pharmacol Sci. 2020 July; 41(7):464-474).The terms “degrader” and “degrader molecule” refer to all compoundscapable of specifically targeting a protein for degradation (e.g.,ATTEC, AUTAC, LYTAC, or PROTAC, reviewed in Ding, et al. 2020).Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emergingalternative therapeutic strategy with the potential to address many ofthe challenges currently faced in modern drug development programs.PROTAC technology employs small molecules that recruit target proteinsfor ubiquitination and removal by the proteasome (see, e.g., Zhou etal., Discovery of a Small-Molecule Degrader of Bromodomain andExtra-Terminal (BET) Proteins with Picomolar Cellular Potencies andCapable of Achieving Tumor Regression. J. Med. Chem. 2018, 61, 462-481;Bondeson and Crews, Targeted Protein Degradation by Small Molecules,Annu Rev Pharmacol Toxicol. 2017 Jan. 6; 57: 107-123; and Lai et al.,Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL AngewChem Int Ed Engl. 2016 Jan 11; 55(2): 807-810). In certain embodiments,LYTACs are particularly advantageous for cell surface proteins asdescribed herein.

Genetic Modifying Agents

In certain embodiments, the one or more modulating agents may be agenetic modifying agent. The genetic modifying agent may comprise aCRISPR system, a zinc finger nuclease system, a TALEN, a meganuclease orRNAi system. In certain embodiments, a target gene is geneticallymodified. In certain embodiments, a target gene RNA is modified, suchthat the modification is temporary. Methods of modifying RNA isdiscussed further herein. In certain embodiments, a gene required forendogenous opioid signaling is targeted. In certain embodiments, genesare modified in cells ex vivo (e.g., NRP1, CRTAM).

CRISPR-Cas Modification

In some embodiments, a polynucleotide of the present invention describedelsewhere herein can be modified using a CRISPR-Cas and/or Cas-basedsystem (e.g., genomic DNA or mRNA, preferably, for a disease gene). Thenucleotide sequence may be or encode one or more components of aCRISPR-Cas system. For example, the nucleotide sequences may be orencode guide RNAs. The nucleotide sequences may also encode CRISPRproteins, variants thereof, or fragments thereof.

In general, a CRISPR-Cas or CRISPR system as used herein and in otherdocuments, such as WO 2014/093622 (PCT/US2013/074667), referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, a tracr(trans-activating CRISPR) sequence (e.g., tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g., CRISPR RNAand transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). See, e.g., Shmakov et al. (2015) “Discovery and FunctionalCharacterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell,DOI: dx.doi.org/10.1016/j.molce1.2015.10.008.

CRISPR-Cas systems can generally fall into two classes based on theirarchitectures of their effector molecules, which are each furthersubdivided by type and subtype. The two class are Class 1 and Class 2.Class 1 CRISPR-Cas systems have effector modules composed of multipleCas proteins, some of which form crRNA-binding complexes, while Class 2CRISPR-Cas systems include a single, multi-domain crRNA-binding protein.

In some embodiments, the CRISPR-Cas system that can be used to modify apolynucleotide of the present invention described herein can be a Class1 CRISPR-Cas system. In some embodiments, the CRISPR-Cas system that canbe used to modify a polynucleotide of the present invention describedherein can be a Class 2 CRISPR-Cas system.

Class 1 CRISPR-Cas Systems

In some embodiments, the CRISPR-Cas system that can be used to modify apolynucleotide of the present invention described herein can be a Class1 CRISPR-Cas system. Class 1 CRISPR-Cas systems are divided into typesI, II, and IV. Makarova et al. 2020. Nat. Rev. 18: 67-83., particularlyas described in FIG. 1. Type I CRISPR-Cas systems are divided into 9subtypes (I-A, I-B, I-C, I-D, I-E, I-F1, I-F2, I-F3, and IG). Makarovaet al., 2020. Class 1, Type I CRISPR-Cas systems can contain a Cas3protein that can have helicase activity. Type III CRISPR-Cas systems aredivided into 6 subtypes (III-A, III-B, III-E, and III-F). Type IIICRISPR-Cas systems can contain a Cas10 that can include an RNArecognition motif called Palm and a cyclase domain that can cleavepolynucleotides. Makarova et al., 2020. Type IV CRISPR-Cas systems aredivided into 3 subtypes. (IV-A, IV-B, and IV-C). .Makarova et al., 2020.Class 1 systems also include CRISPR-Cas variants, including Type I-A,I-B, I-E, I-F and I-U variants, which can include variants carried bytransposons and plasmids, including versions of subtype I-F encoded by alarge family of Tn7-like transposon and smaller groups of Tn7-liketransposons that encode similarly degraded subtype I-B systems. Peterset al., PNAS 114 (35) (2017); DOI: 10.1073/pnas.1709035114; see also,Makarova et al. 2018. The CRISPR Journal, v. 1 , n5, FIG. 5.

The Class 1 systems typically use a multi-protein effector complex,which can, in some embodiments, include ancillary proteins, such as oneor more proteins in a complex referred to as a CRISPR-associated complexfor antiviral defense (Cascade), one or more adaptation proteins (e.g.,Casl, Cas2, RNA nuclease), and/or one or more accessory proteins (e.g.,Cas 4, DNA nuclease), CRISPR associated Rossman fold (CARF) domaincontaining proteins, and/or RNA transcriptase.

The backbone of the Class 1 CRISPR-Cas system effector complexes can beformed by RNA recognition motif domain-containing protein(s) of therepeat-associated mysterious proteins (RAIVIPs) family subunits (e.g.,Cas 5, Cas6, and/or Cas7). RAMP proteins are characterized by having oneor more RNA recognition motif domains. In some embodiments, multiplecopies of RAIVIPs can be present. In some embodiments, the Class ICRISPR-Cas system can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ormore Cas5, Cas6, and/or Cas 7 proteins. In some embodiments, the Cas6protein is an RNAse, which can be responsible for pre-crRNA processing.When present in a Class 1 CRISPR-Cas system, Cas6 can be optionallyphysically associated with the effector complex.

Class 1 CRISPR-Cas system effector complexes can, in some embodiments,also include a large subunit. The large subunit can be composed of orinclude a Cas8 and/or Cas10 protein. See, e.g., FIGS. 1 and 2. Koonin EV, Makarova K S. 2019. Phil. Trans. R. Soc. B 374: 20180087, DOI:10.1098/rstb.2018.0087 and Makarova et al. 2020.

Class 1 CRISPR-Cas system effector complexes can, in some embodiments,include a small subunit (for example, Cash 1). See, e.g., FIGS. 1 and 2.Koonin E V, Makarova K S. 2019 Origins and Evolution of CRISPR-Cassystems. Phil. Trans. R. Soc. B 374: 20180087, DOI:10.1098/rstb.2018.0087.

In some embodiments, the Class 1 CRISPR-Cas system can be a Type ICRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system canbe a subtype I-A CRISPR-Cas system. In some embodiments, the Type ICRISPR-Cas system can be a subtype I-B CRISPR-Cas system. In someembodiments, the Type I CRISPR-Cas system can be a subtype I-CCRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system canbe a subtype I-D CRISPR-Cas system. In some embodiments, the Type ICRISPR-Cas system can be a subtype I-E CRISPR-Cas system. In someembodiments, the Type I CRISPR-Cas system can be a subtype I-F1CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system canbe a subtype I-F2 CRISPR-Cas system. In some embodiments, the Type ICRISPR-Cas system can be a subtype I-F3 CRISPR-Cas system. In someembodiments, the Type I CRISPR-Cas system can be a subtype I-GCRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system canbe a CRISPR Cas variant, such as a Type I-A, I-B, I-E, I-F and I-Uvariants, which can include variants carried by transposons andplasmids, including versions of subtype I-F encoded by a large family ofTn7-like transposon and smaller groups of Tn7-like transposons thatencode similarly degraded subtype I-B systems as previously described.

In some embodiments, the Class 1 CRISPR-Cas system can be a Type IIICRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas systemcan be a subtype III-A CRISPR-Cas system. In some embodiments, the TypeIII CRISPR-Cas system can be a subtype III-B CRISPR-Cas system. In someembodiments, the Type III CRISPR-Cas system can be a subtype III-CCRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas systemcan be a subtype III-D CRISPR-Cas system. In some embodiments, the TypeIII CRISPR-Cas system can be a subtype III-E CRISPR-Cas system. In someembodiments, the Type III CRISPR-Cas system can be a subtype III-FCRISPR-Cas system.

In some embodiments, the Class 1 CRISPR-Cas system can be a Type IVCRISPR-Cas-system. In some embodiments, the Type IV CRISPR-Cas systemcan be a subtype IV-A CRISPR-Cas system. In some embodiments, the TypeIV CRISPR-Cas system can be a subtype IV-B CRISPR-Cas system. In someembodiments, the Type IV CRISPR-Cas system can be a subtype IV-CCRISPR-Cas system.

The effector complex of a Class 1 CRISPR-Cas system can, in someembodiments, include a Cas3 protein that is optionally fused to a Cas2protein, a Cas4, a Cas5, a Cash, a Cas7, a Cas8, a Cas10, a Cas11, or acombination thereof. In some embodiments, the effector complex of aClass 1 CRISPR-Cas system can have multiple copies, such as 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, of any one or more Cas proteins.

Class 2 CRISPR-Cas Systems

The compositions, systems, and methods described in greater detailelsewhere herein can be designed and adapted for use with Class 2CRISPR-Cas systems. Thus, in some embodiments, the CRISPR-Cas system isa Class 2 CRISPR-Cas system. Class 2 systems are distinguished fromClass 1 systems in that they have a single, large, multi-domain effectorprotein. In certain example embodiments, the Class 2 system can be aType II, Type V, or Type VI system, which are described in Makarova etal. “Evolutionary classification of CRISPR-Cas systems: a burst of class2 and derived variants” Nature Reviews Microbiology, 18:67-81 (February2020), incorporated herein by reference. Each type of Class 2 system isfurther divided into subtypes. See Markova et al. 2020, particularly atFIG. 2. Class 2, Type II systems can be divided into 4 subtypes: II-A,II-B, II-C1, and II-C2. Class 2, Type V systems can be divided into 17subtypes: V-A, V-B1, V-B2, V-C, V-D, V-E, V-F1, V-F1(V-U3), V-F2, V-F3,V-G, V-H, V-I, V-K (V-U5), V-U1, V-U2, and V-U4. Class 2, Type IVsystems can be divided into 5 subtypes: VI-A, VI-B1, VI-B2, VI-C, andVI-D.

The distinguishing feature of these types is that their effectorcomplexes consist of a single, large, multi-domain protein. Type Vsystems differ from Type II effectors (e.g., Cas9), which contain twonuclear domains that are each responsible for the cleavage of one strandof the target DNA, with the HNH nuclease inserted inside the Ruv-C likenuclease domain sequence. The Type V systems (e.g., Cas12) only containa RuvC-like nuclease domain that cleaves both strands. Type VI (Cas13)are unrelated to the effectors of Type II and V systems and contain twoHEPN domains and target RNA. Cas13 proteins also display collateralactivity that is triggered by target recognition. Some Type V systemshave also been found to possess this collateral activity with twosingle-stranded DNA in in vitro contexts.

In some embodiments, the Class 2 system is a Type II system. In someembodiments, the Type II CRISPR-Cas system is a II-A CRISPR-Cas system.In some embodiments, the Type II CRISPR-Cas system is a II-B CRISPR-Cassystem. In some embodiments, the Type II CRISPR-Cas system is a II-C1CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system isa II-C2 CRISPR-Cas system. In some embodiments, the Type II system is aCas9 system. In some embodiments, the Type II system includes a Cas9.

In some embodiments, the Class 2 system is a Type V system. In someembodiments, the Type V CRISPR-Cas system is a V-A CRISPR-Cas system. Insome embodiments, the Type V CRISPR-Cas system is a V-B 1 CRISPR-Cassystem. In some embodiments, the Type V CRISPR-Cas system is a V-B2CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system isa V-C CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cassystem is a V-D CRISPR-Cas system. In some embodiments, the Type VCRISPR-Cas system is a V-E CRISPR-Cas system. In some embodiments, theType V CRISPR-Cas system is a V-F1 CRISPR-Cas system. In someembodiments, the Type V CRISPR-Cas system is a V-F1 (V-U3) CRISPR-Cassystem. In some embodiments, the Type V CRISPR-Cas system is a V-F2CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system isa V-F3 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cassystem is a V-G CRISPR-Cas system. In some embodiments, the Type VCRISPR-Cas system is a V-H CRISPR-Cas system. In some embodiments, theType V CRISPR-Cas system is a V-I CRISPR-Cas system. In someembodiments, the Type V CRISPR-Cas system is a V-K (V-U5) CRISPR-Cassystem. In some embodiments, the Type V CRISPR-Cas system is a V-U1CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system isa V-U2 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cassystem is a V-U4 CRISPR-Cas system. In some embodiments, the Type VCRISPR-Cas system includes a Cas12a (Cpf1), Cas12b (C2c1), Cas12c(C2c3), CasX, and/or Cas14.

In some embodiments the Class 2 system is a Type VI system. In someembodiments, the Type VI CRISPR-Cas system is a VI-A CRISPR-Cas system.In some embodiments, the Type VI CRISPR-Cas system is a VI-B1 CRISPR-Cassystem. In some embodiments, the Type VI CRISPR-Cas system is a VI-B2CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system isa VI-C CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cassystem is a VI-D CRISPR-Cas system. In some embodiments, the Type VICRISPR-Cas system includes a Cas13a (C2c2), Cas13b (Group 29/30),Cas13c, and/or Cas13d.

Specialized Cas-Based Systems

In some embodiments, the system is a Cas-based system that is capable ofperforming a specialized function or activity. For example, the Casprotein may be fused, operably coupled to, or otherwise associated withone or more functionals domains. In certain example embodiments, the Casprotein may be a catalytically dead Cas protein (“dCas”) and/or havenickase activity. A nickase is a Cas protein that cuts only one strandof a double stranded target. In such embodiments, the dCas or nickaseprovide a sequence specific targeting functionality that delivers thefunctional domain to or proximate a target sequence. Example functionaldomains that may be fused to, operably coupled to, or otherwiseassociated with a Cas protein can be or include, but are not limited toa nuclear localization signal (NLS) domain, a nuclear export signal(NES) domain, a translational activation domain, a transcriptionalactivation domain (e.g. VP64, p65, MyoD1, HSF1, RTA, and SETT/9), atranslation initiation domain, a transcriptional repression domain(e.g., a KRAB domain, NuE domain, NcoR domain, and a SID domain such asa SID4X domain), a nuclease domain (e.g., Fold), a histone modificationdomain (e.g., a histone acetyltransferase), a lightinducible/controllable domain, a chemically inducible/controllabledomain, a transposase domain, a homologous recombination machinerydomain, a recombinase domain, an integrase domain, and combinationsthereof. Methods for generating catalytically dead Cas9 or a nickaseCas9 (WO 2014/204725, Ran et al. Cell. 2013 Sep. 12; 154(6):1380-1389),Cas12 (Liu et al. Nature Communications, 8, 2095 (2017) , and Cas13 (WO2019/005884, WO2019/060746) are known in the art and incorporated hereinby reference.

In some embodiments, the functional domains can have one or more of thefollowing activities: methylase activity, demethylase activity,translation activation activity, translation initiation activity,translation repression activity, transcription activation activity,transcription repression activity, transcription release factoractivity, histone modification activity, nuclease activity,single-strand RNA cleavage activity, double-strand RNA cleavageactivity, single-strand DNA cleavage activity, double-strand DNAcleavage activity, molecular switch activity, chemical inducibility,light inducibility, and nucleic acid binding activity. In someembodiments, the one or more functional domains may comprise epitopetags or reporters. Non-limiting examples of epitope tags includehistidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA)tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples ofreporters include, but are not limited to, glutathione-S-transferase(GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase(CAT) beta-galactosidase, beta-glucuronidase, luciferase, greenfluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP),yellow fluorescent protein (YFP), and auto-fluorescent proteinsincluding blue fluorescent protein (BFP).

The one or more functional domain(s) may be positioned at, near, and/orin proximity to a terminus of the effector protein (e.g., a Casprotein). In embodiments having two or more functional domains, each ofthe two can be positioned at or near or in proximity to a terminus ofthe effector protein (e.g., a Cas protein). In some embodiments, such asthose where the functional domain is operably coupled to the effectorprotein, the one or more functional domains can be tethered or linkedvia a suitable linker (including, but not limited to, GlySer linkers) tothe effector protein (e.g., a Cas protein). When there is more than onefunctional domain, the functional domains can be same or different. Insome embodiments, all the functional domains are the same. In someembodiments, all of the functional domains are different from eachother. In some embodiments, at least two of the functional domains aredifferent from each other. In some embodiments, at least two of thefunctional domains are the same as each other.

Other suitable functional domains can be found, for example, inInternational Application Publication No. WO 2019/018423.

Split CRISPR-Cas Systems

In some embodiments, the CRISPR-Cas system is a split CRISPR-Cas system.See e.g., Zetche et al., 2015. Nat. Biotechnol. 33(2): 139-142 and WO2019/018423 , the compositions and techniques of which can be used inand/or adapted for use with the present invention. Split CRISPR-Casproteins are set forth herein and in documents incorporated herein byreference in further detail herein. In certain embodiments, each part ofa split CRISPR protein are attached to a member of a specific bindingpair, and when bound with each other, the members of the specificbinding pair maintain the parts of the CRISPR protein in proximity. Incertain embodiments, each part of a split CRISPR protein is associatedwith an inducible binding pair. An inducible binding pair is one whichis capable of being switched “on” or “off” by a protein or smallmolecule that binds to both members of the inducible binding pair. Insome embodiments, CRISPR proteins may preferably split between domains,leaving domains intact. In particular embodiments, said Cas splitdomains (e.g., RuvC and HNH domains in the case of Cas9) can besimultaneously or sequentially introduced into the cell such that saidsplit Cas domain(s) process the target nucleic acid sequence in thealgae cell. The reduced size of the split Cas compared to the wild typeCas allows other methods of delivery of the systems to the cells, suchas the use of cell penetrating peptides as described herein.

DNA and RNA Base Editing

In some embodiments, a polynucleotide of the present invention describedelsewhere herein can be modified using a base editing system. In someembodiments, a Cas protein is connected or fused to a nucleotidedeaminase. Thus, in some embodiments the Cas-based system can be a baseediting system. As used herein “base editing” refers generally to theprocess of polynucleotide modification via a CRISPR-Cas-based orCas-based system that does not include excising nucleotides to make themodification. Base editing can convert base pairs at precise locationswithout generating excess undesired editing byproducts that can be madeusing traditional CRISPR-Cas systems.

In certain example embodiments, the nucleotide deaminase may be a DNAbase editor used in combination with a DNA binding Cas protein such as,but not limited to, Class 2 Type II and Type V systems. Two classes ofDNA base editors are generally known: cytosine base editors (CBEs) andadenine base editors (ABEs). CBEs convert a C•G base pair into a T•Abase pair (Komor et al. 2016. Nature. 533:420-424; Nishida et al. 2016.Science. 353; and Li et al. Nat. Biotech. 36:324-327) and ABEs convertan A•T base pair to a G•C base pair. Collectively, CBEs and ABEs canmediate all four possible transition mutations (C to T, A to G, T to C,and G to A). Rees and Liu. 2018.Nat. Rev. Genet. 19(12): 770-788,particularly at FIGS. 1 b, 2 a-2 c, 3 a-3 f, and Table 1. In someembodiments, the base editing system includes a CBE and/or an ABE. Insome embodiments, a polynucleotide of the present invention describedelsewhere herein can be modified using a base editing system. Rees andLiu. 2018. Nat. Rev. Gent. 19(12):770-788. Base editors also generallydo not need a DNA donor template and/or rely on homology-directedrepair. Komor et al. 2016. Nature. 533:420-424; Nishida et al. 2016.Science. 353; and Gaudeli et al. 2017. Nature. 551:464-471. Upon bindingto a target locus in the DNA, base pairing between the guide RNA of thesystem and the target DNA strand leads to displacement of a smallsegment of ssDNA in an “R-loop”. Nishimasu et al. Cell. 156:935-949. DNAbases within the ssDNA bubble are modified by the enzyme component, suchas a deaminase. In some systems, the catalytically disabled Cas proteincan be a variant or modified Cas can have nickase functionality and cangenerate a nick in the non-edited DNA strand to induce cells to repairthe non-edited strand using the edited strand as a template. Komor etal. 2016. Nature. 533:420-424; Nishida et al. 2016. Science. 353; andGaudeli et al. 2017. Nature. 551:464-471. Base editors may be furtherengineered to optimize conversion of nucleotides (e.g. A:T to G:C).Richter et al. 2020. Nature Biotechnology.doi.org/10.1038/s41587-020-0453-z.

Other Example Type V base editing systems are described in WO2018/213708, WO 2018/213726, PCT/US2018/067207, PCT/US2018/067225, andPCT/US2018/067307 which are incorporated by referenced herein.

In certain example embodiments, the base editing system may be a RNAbase editing system. As with DNA base editors, a nucleotide deaminasecapable of converting nucleotide bases may be fused to a Cas protein.However, in these embodiments, the Cas protein will need to be capableof binding RNA. Example RNA binding Cas proteins include, but are notlimited to, RNA-binding Cas9s such as Francisella novicida Cas9(“FnCas9”), and Class 2 Type VI Cas systems. The nucleotide deaminasemay be a cytidine deaminase or an adenosine deaminase, or an adenosinedeaminase engineered to have cytidine deaminase activity. In certainexample embodiments, the RNA based editor may be used to delete orintroduce a post-translation modification site in the expressed mRNA. Incontrast to DNA base editors, whose edits are permanent in the modifiedcell, RNA base editors can provide edits where finer temporal controlmay be needed, for example in modulating a particular immune response.Example Type VI RNA-base editing systems are described in Cox et al.2017. Science 358: 1019-1027, WO 2019/005884, WO 2019/005886, WO2019/071048, PCT/U520018/05179, PCT/US2018/067207, which areincorporated herein by reference. An example FnCas9 system that may beadapted for RNA base editing purposes is described in WO 2016/106236,which is incorporated herein by reference.

An example method for delivery of base-editing systems, including use ofa split-intein approach to divide CBE and ABE into reconstituble halves,is described in Levy et al. Nature Biomedical Engineeringdoi.org/10.1038/s41441-019-0505-5 (2019), which is incorporated hereinby reference.

Prime Editors

In some embodiments, a polynucleotide of the present invention describedelsewhere herein can be modified using a prime editing system (See e.g.Anzalone et al. 2019. Nature. 576: 149-157). Like base editing systems,prime editing systems can be capable of targeted modification of apolynucleotide without generating double stranded breaks and does notrequire donor templates. Further prime editing systems can be capable ofall 12 possible combination swaps. Prime editing can operate via a“search-and-replace” methodology and can mediate targeted insertions,deletions, all 12 possible base-to-base conversion, and combinationsthereof. Generally, a prime editing system, as exemplified by PE1, PE2,and PE3 (Id.), can include a reverse transcriptase fused or otherwisecoupled or associated with an RNA-programmable nickase, and aprime-editing extended guide RNA (pegRNA) to facility direct copying ofgenetic information from the extension on the pegRNA into the targetpolynucleotide. Embodiments that can be used with the present inventioninclude these and variants thereof. Prime editing can have the advantageof lower off-target activity than traditional CRIPSR-Cas systems alongwith few byproducts and greater or similar efficiency as compared totraditional CRISPR-Cas systems.

In some embodiments, the prime editing guide molecule can specify boththe target polynucleotide information (e.g. sequence) and contain a newpolynucleotide cargo that replaces target polynucleotides. To initiatetransfer from the guide molecule to the target polynucleotide, the PEsystem can nick the target polynucleotide at a target side to expose a3′hydroxyl group, which can prime reverse transcription of anedit-encoding extension region of the guide molecule (e.g. a primeediting guide molecule or peg guide molecule) directly into the targetsite in the target polynucleotide. See e.g. Anzalone et al. 2019.Nature. 576: 149-157, particularly at FIGS. 1b, 1c , related discussion,and Supplementary discussion.

In some embodiments, a prime editing system can be composed of a Caspolypeptide having nickase activity, a reverse transcriptase, and aguide molecule. The Cas polypeptide can lack nuclease activity. Theguide molecule can include a target binding sequence as well as a primerbinding sequence and a template containing the edited polynucleotidesequence. The guide molecule, Cas polypeptide, and/or reversetranscriptase can be coupled together or otherwise associate with eachother to form an effector complex and edit a target sequence. In someembodiments, the Cas polypeptide is a Class 2, Type V Cas polypeptide.In some embodiments, the Cas polypeptide is a Cas9 polypeptide (e.g. isa Cas9 nickase). In some embodiments, the Cas polypeptide is fused tothe reverse transcriptase. In some embodiments, the Cas polypeptide islinked to the reverse transcriptase.

In some embodiments, the prime editing system can be a PE1 system orvariant thereof, a PE2 system or variant thereof, or a PE3 (e.g. PE3,PE3b) system. See e.g., Anzalone et al. 2019. Nature. 576: 149-157,particularly at pgs. 2-3, FIGS. 2a, 3a -3 f, 4 a-4 b, Extended dataFIGS. 3a -3 b, 4,

The peg guide molecule can be about 10 to about 200 or more nucleotidesin length, such as 10 to/or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, or 200 or more nucleotides in length.Optimization of the peg guide molecule can be accomplished as describedin Anzalone et al. 2019. Nature. 576: 149-157, particularly at pg. 3,FIG. 2a -2 b, and Extended Data FIGS. 5a -c.

CRISPR Associated Transposase (CAST) Systems

In some embodiments, a polynucleotide of the present invention describedelsewhere herein can be modified using a CRISPR Associated Transposase(“CAST”) system. CAST system can include a Cas protein that iscatalytically inactive, or engineered to be catalytically active, andfurther comprises a transposase (or subunits thereof) that catalyzeRNA-guided DNA transposition. Such systems are able to insert DNAsequences at a target site in a DNA molecule without relying on hostcell repair machinery. CAST systems can be Classl or Class 2 CASTsystems. An example Class 1 system is described in Klompe et al. Nature,doi:10.1038/s41586-019-1323, which is in incorporated herein byreference. An example Class 2 system is described in Strecker et al.Science. 10/1126/science. aax9181 (2019), and PCT/US2019/066835 whichare incorporated herein by reference.

Guide Molecules

The CRISPR-Cas or Cas-Based system described herein can, in someembodiments, include one or more guide molecules. The terms guidemolecule, guide sequence and guide polynucleotide, refer topolynucleotides capable of guiding Cas to a target genomic locus and areused interchangeably as in foregoing cited documents such as WO2014/093622 (PCT/US2013/074667). In general, a guide sequence is anypolynucleotide sequence having sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence.The guide molecule can be a polynucleotide.

The ability of a guide sequence (within a nucleic acid-targeting guideRNA) to direct sequence-specific binding of a nucleic acid-targetingcomplex to a target nucleic acid sequence may be assessed by anysuitable assay. For example, the components of a nucleic acid-targetingCRISPR system sufficient to form a nucleic acid-targeting complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target nucleic acid sequence, such as bytransfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay (Qui et al. 2004. BioTechniques. 36(4)702-707).Similarly, cleavage of a target nucleic acid sequence may be evaluatedin a test tube by providing the target nucleic acid sequence, componentsof a nucleic acid-targeting complex, including the guide sequence to betested and a control guide sequence different from the test guidesequence, and comparing binding or rate of cleavage at the targetsequence between the test and control guide sequence reactions. Otherassays are possible and will occur to those skilled in the art.

In some embodiments, the guide molecule is an RNA. The guide molecule(s)(also referred to interchangeably herein as guide polynucleotide andguide sequence) that are included in the CRISPR-Cas or Cas based systemcan be any polynucleotide sequence having sufficient complementaritywith a target nucleic acid sequence to hybridize with the target nucleicacid sequence and direct sequence-specific binding of a nucleicacid-targeting complex to the target nucleic acid sequence. In someembodiments, the degree of complementarity, when optimally aligned usinga suitable alignment algorithm, can be about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences, non-limiting examples of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq. sourceforge. net).

A guide sequence, and hence a nucleic acid-targeting guide, may beselected to target any target nucleic acid sequence. The target sequencemay be DNA. The target sequence may be any RNA sequence. In someembodiments, the target sequence may be a sequence within an RNAmolecule selected from the group consisting of messenger RNA (mRNA),pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA),small interfering RNA (siRNA), small nuclear RNA (snRNA), smallnucleolar RNA (snoRNA), double stranded RNA (dsRNA), non-coding RNA(ncRNA), long non-coding RNA (lncRNA), and small cytoplasmatic RNA(scRNA). In some preferred embodiments, the target sequence may be asequence within an RNA molecule selected from the group consisting ofmRNA, pre-mRNA, and rRNA. In some preferred embodiments, the targetsequence may be a sequence within an RNA molecule selected from thegroup consisting of ncRNA, and lncRNA. In some more preferredembodiments, the target sequence may be a sequence within an mRNAmolecule or a pre-mRNA molecule.

In some embodiments, a nucleic acid-targeting guide is selected toreduce the degree secondary structure within the nucleic acid-targetingguide. In some embodiments, about or less than about 75%, 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleicacid-targeting guide participate in self-complementary base pairing whenoptimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and P A Carrand G M Church, 2009, Nature Biotechnology 27(12): 1151-62).

In certain embodiments, a guide RNA or crRNA may comprise, consistessentially of, or consist of a direct repeat (DR) sequence and a guidesequence or spacer sequence. In certain embodiments, the guide RNA orcrRNA may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.In certain embodiments, the direct repeat sequence may be locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In otherembodiments, the direct repeat sequence may be located downstream (i.e.,3′) from the guide sequence or spacer sequence.

In certain embodiments, the crRNA comprises a stem loop, preferably asingle stem loop. In certain embodiments, the direct repeat sequenceforms a stem loop, preferably a single stem loop.

In certain embodiments, the spacer length of the guide RNA is from 15 to35 nt. In certain embodiments, the spacer length of the guide RNA is atleast 15 nucleotides. In certain embodiments, the spacer length is from15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19,or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27nt, from 27 to 30 nt, e.g., 27, 28, 29, or 30 nt, from 30 to 35 nt,e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.

The “tracrRNA” sequence or analogous terms includes any polynucleotidesequence that has sufficient complementarity with a crRNA sequence tohybridize. In some embodiments, the degree of complementarity betweenthe tracrRNA sequence and crRNA sequence along the length of the shorterof the two when optimally aligned is about or more than about 25%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In someembodiments, the tracr sequence is about or more than about 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or morenucleotides in length. In some embodiments, the tracr sequence and crRNAsequence are contained within a single transcript, such thathybridization between the two produces a transcript having a secondarystructure, such as a hairpin.

In general, degree of complementarity is with reference to the optimalalignment of the sca sequence and tracr sequence, along the length ofthe shorter of the two sequences. Optimal alignment may be determined byany suitable alignment algorithm, and may further account for secondarystructures, such as self-complementarity within either the sca sequenceor tracr sequence. In some embodiments, the degree of complementaritybetween the tracr sequence and sca sequence along the length of theshorter of the two when optimally aligned is about or more than about25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.

In some embodiments, the degree of complementarity between a guidesequence and its corresponding target sequence can be about or more thanabout 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%; a guide orRNA or sgRNA can be about or more than about 5, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 75, or more nucleotides in length; or guide or RNA or sgRNA can beless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length; and tracr RNA can be 30 or 50 nucleotides inlength. In some embodiments, the degree of complementarity between aguide sequence and its corresponding target sequence is greater than94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or99% or 99.5% or 99.9%, or 100%. Off target is less than 100% or 99.9% or99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88%or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementaritybetween the sequence and the guide, with it advantageous that off targetis 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97%or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between thesequence and the guide.

In some embodiments according to the invention, the guide RNA (capableof guiding Cas to a target locus) may comprise (1) a guide sequencecapable of hybridizing to a genomic target locus in the eukaryotic cell;(2) a tracr sequence; and (3) a tracr mate sequence. All (1) to (3) mayreside in a single RNA, i.e., an sgRNA (arranged in a 5′ to 3′orientation), or the tracr RNA may be a different RNA than the RNAcontaining the guide and tracr sequence. The tracr hybridizes to thetracr mate sequence and directs the CRISPR/Cas complex to the targetsequence. Where the tracr RNA is on a different RNA than the RNAcontaining the guide and tracr sequence, the length of each RNA may beoptimized to be shortened from their respective native lengths, and eachmay be independently chemically modified to protect from degradation bycellular RNase or otherwise increase stability.

Many modifications to guide sequences are known in the art and arefurther contemplated within the context of this invention. Variousmodifications may be used to increase the specificity of binding to thetarget sequence and/or increase the activity of the Cas protein and/orreduce off-target effects. Example guide sequence modifications aredescribed in PCT US2019/045582, specifically paragraphs [0178]-[0333].which is incorporated herein by reference.

Target Sequences, PAMs, and PFSs Target Sequences

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise RNA polynucleotides. The term “target RNA” refersto an RNA polynucleotide being or comprising the target sequence. Inother words, the target polynucleotide can be a polynucleotide or a partof a polynucleotide to which a part of the guide sequence is designed tohave complementarity with and to which the effector function mediated bythe complex comprising the CRISPR effector protein and a guide moleculeis to be directed. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell.

The guide sequence can specifically bind a target sequence in a targetpolynucleotide. The target polynucleotide may be DNA. The targetpolynucleotide may be RNA. The target polynucleotide can have one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. or more) targetsequences. The target polynucleotide can be on a vector. The targetpolynucleotide can be genomic DNA. The target polynucleotide can beepisomal. Other forms of the target polynucleotide are describedelsewhere herein.

The target sequence may be DNA. The target sequence may be any RNAsequence. In some embodiments, the target sequence may be a sequencewithin an RNA molecule selected from the group consisting of messengerRNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA),micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA(snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA),non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and smallcytoplasmatic RNA (scRNA). In some preferred embodiments, the targetsequence (also referred to herein as a target polynucleotide) may be asequence within an RNA molecule selected from the group consisting ofmRNA, pre-mRNA, and rRNA. In some preferred embodiments, the targetsequence may be a sequence within an RNA molecule selected from thegroup consisting of ncRNA, and lncRNA. In some more preferredembodiments, the target sequence may be a sequence within an mRNAmolecule or a pre-mRNA molecule.

PAM and PFS Elements

PAM elements are sequences that can be recognized and bound by Casproteins. Cas proteins/effector complexes can then unwind the dsDNA at aposition adjacent to the PAM element. It will be appreciated that Casproteins and systems that include them that target RNA do not requirePAM sequences (Marraffini et al. 2010. Nature. 463:568-571). Instead,many rely on PF Ss, which are discussed elsewhere herein. In certainembodiments, the target sequence should be associated with a PAM(protospacer adjacent motif) or PFS (protospacer flanking sequence orsite), that is, a short sequence recognized by the CRISPR complex.Depending on the nature of the CRISPR-Cas protein, the target sequenceshould be selected, such that its complementary sequence in the DNAduplex (also referred to herein as the non-target sequence) is upstreamor downstream of the PAM. In the embodiments, the complementary sequenceof the target sequence is downstream or 3′ of the PAM or upstream or 5′of the PAM. The precise sequence and length requirements for the PAMdiffer depending on the Cas protein used, but PAMs are typically 2-5base pair sequences adjacent the protospacer (that is, the targetsequence). Examples of the natural PAM sequences for different Casproteins are provided herein below and the skilled person will be ableto identify further PAM sequences for use with a given Cas protein.

The ability to recognize different PAM sequences depends on the Caspolypeptide(s) included in the system. See e.g., Gleditzsch et al. 2019.RNA Biology. 16(4):504-517. Table 2 below shows several Cas polypeptidesand the PAM sequence they recognize.

TABLE 2 Example PAM Sequences Cas Protein PAM Sequence SpCas9 NGG/NRGSaCas9 NGRRT (SEQ ID NO: 1) or NGRRN (SEQ ID NO: 2) NmeCas9NNNNGATT (SEQ ID NO: 3) CjCas9 NNNNRYAC (SEQ ID NO: 4) StCas9NNAGAAW (SEQ ID NO: 5) Cas12a (Cpf1) (including  TTTV (SEQ ID NO: 6)LbCpf1 and AsCpf1) Cas12b (C2c1) TTT, TTA, and TTC Cas12c (C2c3) TACas12d (CasY) TA Cas12e (CasX) 5′-TTCN-3′ (SEQ ID NO: 7)

In a preferred embodiment, the CRISPR effector protein may recognize a3′ PAM. In certain embodiments, the CRISPR effector protein mayrecognize a 3′ PAM which is 5′H, wherein H is A, C or U.

Further, engineering of the PAM Interacting (PI) domain on the Casprotein may allow programing of PAM specificity, improve target siterecognition fidelity, and increase the versatility of the CRISPR-Casprotein, for example as described for Cas9 in Kleinstiver BP et al.Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature.2015 Jul. 23; 523(7561):481-5. doi: 10.1038/nature14592. As furtherdetailed herein, the skilled person will understand that Cas13 proteinsmay be modified analogously. Gao et al, “Engineered Cpfl Enzymes withAltered PAM Specificities,” bioRxiv 091611; doi:http://dx.doi.org/10.1101/091611 (Dec. 4, 2016). Doench et al. created apool of sgRNAs, tiling across all possible target sites of a panel ofsix endogenous mouse and three endogenous human genes and quantitativelyassessed their ability to produce null alleles of their target gene byantibody staining and flow cytometry. The authors showed thatoptimization of the PAM improved activity and also provided an on-linetool for designing sgRNAs.

PAM sequences can be identified in a polynucleotide using an appropriatedesign tool, which are commercially available as well as online. Suchfreely available tools include, but are not limited to, CRISPRFinder andCRISPRTarget. Mojica et al. 2009. Microbiol. 155(Pt. 3):733-740; Atschulet al. 1990. J. Mol. Biol. 215:403-410; Biswass et al. 2013 RNA Biol.10:817-827; and Grissa et al. 2007. Nucleic Acid Res. 35:W52-57.Experimental approaches to PAM identification can include, but are notlimited to, plasmid depletion assays (Jiang et al. 2013. Nat.Biotechnol. 31:233-239; Esvelt et al. 2013. Nat. Methods. 10:1116-1121;Kleinstiver et al. 2015. Nature. 523:481-485), screened by ahigh-throughput in vivo model called PAM-SCNAR (Pattanayak et al. 2013.Nat. Biotechnol. 31:839-843 and Leenay et al. 2016.Mol. Cell. 16:253),and negative screening (Zetsche et al. 2015. Cell. 163:759-771).

As previously mentioned, CRISPR-Cas systems that target RNA do nottypically rely on PAM sequences. Instead such systems typicallyrecognize protospacer flanking sites (PFSs) instead of PAMs Thus, TypeVI CRISPR-Cas systems typically recognize protospacer flanking sites(PFSs) instead of PAMs. PFSs represents an analogue to PAMs for RNAtargets. Type VI CRISPR-Cas systems employ a Cas13. Some Cas13 proteinsanalyzed to date, such as Cas13a (C2c2) identified from Leptotrichiashahii (LShCAs13a) have a specific discrimination against G at the 3′end of the target RNA. The presence of a C at the corresponding crRNArepeat site can indicate that nucleotide pairing at this position isrejected. However, some Cas13 proteins (e.g., LwaCAs13a and PspCas13b)do not seem to have a PFS preference. See e.g., Gleditzsch et al. 2019.RNA Biology. 16(4):504-517.

Some Type VI proteins, such as subtype B, have 5′-recognition of D (G,T, A) and a 3′-motif requirement of NAN or NNA. One example is theCas13b protein identified in Bergeyella zoohelcum (BzCas13b). See e.g.,Gleditzsch et al. 2019. RNA Biology. 16(4):504-517.

Overall Type VI CRISPR-Cas systems appear to have less restrictive rulesfor substrate (e.g., target sequence) recognition than those that targetDNA (e.g., Type V and type II).

Zinc Finger Nucleases

In some embodiments, the polynucleotide is modified using a Zinc Fingernuclease or system thereof. One type of programmable DNA-binding domainis provided by artificial zinc-finger (ZF) technology, which involvesarrays of ZF modules to target new DNA-binding sites in the genome. Eachfinger module in a ZF array targets three DNA bases. A customized arrayof individual zinc finger domains is assembled into a ZF protein (ZFP).ZFPs can comprise a functional domain. The first synthetic zinc fingernucleases (ZFNs) were developed by fusing a ZF protein to the catalyticdomain of the Type IIS restriction enzyme Fokl. (Kim, Y. G. et al.,1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. U.S.A.91, 883-887; Kim, Y. G. et al., 1996, Hybrid restriction enzymes: zincfinger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. U.S.A.93, 1156-1160). Increased cleavage specificity can be attained withdecreased off target activity by use of paired ZFN heterodimers, eachtargeting different nucleotide sequences separated by a short spacer.(Doyon, Y. et al., 2011, Enhancing zinc-finger-nuclease activity withimproved obligate heterodimeric architectures. Nat. Methods 8, 74-79).ZFPs can also be designed as transcription activators and repressors andhave been used to target many genes in a wide variety of organisms.Exemplary methods of genome editing using ZFNs can be found for examplein U.S. Pat. Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978,6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719,7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626,all of which are specifically incorporated by reference.

TALE Nucleases

In some embodiments, a TALE nuclease or TALE nuclease system can be usedto modify a polynucleotide. In some embodiments, the methods providedherein use isolated, non-naturally occurring, recombinant or engineeredDNA binding proteins that comprise TALE monomers or TALE monomers orhalf monomers as a part of their organizational structure that enablethe targeting of nucleic acid sequences with improved efficiency andexpanded specificity.

Naturally occurring TALEs or “wild type TALEs” are nucleic acid bindingproteins secreted by numerous species of proteobacteria. TALEpolypeptides contain a nucleic acid binding domain composed of tandemrepeats of highly conserved monomer polypeptides that are predominantly33, 34 or 35 amino acids in length and that differ from each othermainly in amino acid positions 12 and 13. In advantageous embodimentsthe nucleic acid is DNA. As used herein, the term “polypeptidemonomers”, “TALE monomers” or “monomers” will be used to refer to thehighly conserved repetitive polypeptide sequences within the TALEnucleic acid binding domain and the term “repeat variable di-residues”or “RVD” will be used to refer to the highly variable amino acids atpositions 12 and 13 of the polypeptide monomers. As provided throughoutthe disclosure, the amino acid residues of the RVD are depicted usingthe IUPAC single letter code for amino acids. A general representationof a TALE monomer which is comprised within the DNA binding domain isX₁₋₁₁-(X₁₂X₁₃)-X₁₄₋₃₃ or  ₃₄ or  ₃₅, where the subscript indicates theamino acid position and X represents any amino acid. X₁₂X₁₃ indicate theRVDs. In some polypeptide monomers, the variable amino acid at position13 is missing or absent and in such monomers, the RVD consists of asingle amino acid. In such cases the RVD may be alternativelyrepresented as X*, where X represents X₁₂ and (*) indicates that X₁₃ isabsent. The DNA binding domain comprises several repeats of TALEmonomers and this may be represented as (X₁₋₁₁-(X₁₂X₁₃)-X₁₄₋₃₃ or  ₃₄ or ₃₅)_(z), where in an advantageous embodiment, z is at least 5 to 40. Ina further advantageous embodiment, z is at least 10 to 26.

The TALE monomers can have a nucleotide binding affinity that isdetermined by the identity of the amino acids in its RVD. For example,polypeptide monomers with an RVD of NI can preferentially bind toadenine (A), monomers with an RVD of NG can preferentially bind tothymine (T), monomers with an RVD of HD can preferentially bind tocytosine (C) and monomers with an RVD of NN can preferentially bind toboth adenine (A) and guanine (G). In some embodiments, monomers with anRVD of IG can preferentially bind to T. Thus, the number and order ofthe polypeptide monomer repeats in the nucleic acid binding domain of aTALE determines its nucleic acid target specificity. In someembodiments, monomers with an RVD of NS can recognize all four basepairs and can bind to A, T, G or C. The structure and function of TALEsis further described in, for example, Moscou et al., Science 326:1501(2009); Boch et al., Science 326:1509-1512 (2009); and Zhang et al.,Nature Biotechnology 29:149-153 (2011).

The polypeptides used in methods of the invention can be isolated,non-naturally occurring, recombinant or engineered nucleic acid-bindingproteins that have nucleic acid or DNA binding regions containingpolypeptide monomer repeats that are designed to target specific nucleicacid sequences.

As described herein, polypeptide monomers having an RVD of HN or NHpreferentially bind to guanine and thereby allow the generation of TALEpolypeptides with high binding specificity for guanine containing targetnucleic acid sequences. In some embodiments, polypeptide monomers havingRVDs RN, NN, NK, SN, NH, KN, HN, NQ, HH, RG, KH, RH and SS canpreferentially bind to guanine. In some embodiments, polypeptidemonomers having RVDs RN, NK, NQ, HH, KH, RH, SS and SN canpreferentially bind to guanine and can thus allow the generation of TALEpolypeptides with high binding specificity for guanine containing targetnucleic acid sequences. In some embodiments, polypeptide monomers havingRVDs HH, KH, NH, NK, NQ, RH, RN and SS can preferentially bind toguanine and thereby allow the generation of TALE polypeptides with highbinding specificity for guanine containing target nucleic acidsequences. In some embodiments, the RVDs that have high bindingspecificity for guanine are RN, NH RH and KH. Furthermore, polypeptidemonomers having an RVD of NV can preferentially bind to adenine andguanine. In some embodiments, monomers having RVDs of H*, HA, KA, N*,NA, NC, NS, RA, and S* bind to adenine, guanine, cytosine and thyminewith comparable affinity.

The predetermined N-terminal to C-terminal order of the one or morepolypeptide monomers of the nucleic acid or DNA binding domaindetermines the corresponding predetermined target nucleic acid sequenceto which the polypeptides of the invention will bind. As used herein themonomers and at least one or more half monomers are “specificallyordered to target” the genomic locus or gene of interest. In plantgenomes, the natural TALE-binding sites always begin with a thymine (T),which may be specified by a cryptic signal within the non-repetitiveN-terminus of the TALE polypeptide; in some cases, this region may bereferred to as repeat 0. In animal genomes, TALE binding sites do notnecessarily have to begin with a thymine (T) and polypeptides of theinvention may target DNA sequences that begin with T, A, G or C. Thetandem repeat of TALE monomers always ends with a half-length repeat ora stretch of sequence that may share identity with only the first 20amino acids of a repetitive full-length TALE monomer and this halfrepeat may be referred to as a half-monomer. Therefore, it follows thatthe length of the nucleic acid or DNA being targeted is equal to thenumber of full monomers plus two.

As described in Zhang et al., Nature Biotechnology 29:149-153 (2011),TALE polypeptide binding efficiency may be increased by including aminoacid sequences from the “capping regions” that are directly N-terminalor C-terminal of the DNA binding region of naturally occurring TALEsinto the engineered TALEs at positions N-terminal or C-terminal of theengineered TALE DNA binding region. Thus, in certain embodiments, theTALE polypeptides described herein further comprise an N-terminalcapping region and/or a C-terminal capping region.

An exemplary amino acid sequence of a N-terminal capping region is:

(SEQ ID NO: 8) M D P I R S R T P S P A R E L L S G P Q P D G VQ P T A D R G V S P P A G G P L D G L P A R R TM S R T R L P S P P A P S P A F S A D S F S D LL R Q F D P S L F N T S L F D S L P P F G A H HT E A A T G E W D E V Q S G L R A A D A P P P TM R V A V T A A R P P R A K P A P R R R A A Q PS D A S P A A Q V D L R T L G Y S Q Q Q Q E K IK P K V R S T V A Q H H E A L V G H G F T H A HI V A L S Q H P A A L G T V A V K Y Q D M I A AL P E A T H E A I V G V G K Q W S G A R A L E AL L T V A G E L R G P P L Q L D T G Q L L K I AK R G G V T A V E A V H A W R N A L T G A P L N

An exemplary amino acid sequence of a C-terminal capping region is:

(SEQ ID NO: 9) R P A L E S I V A Q L S R P D P A L A A L T N DH L V A L A C L G G R P A L D A V K K G L P H AP A L I K R T N R R I P E R T S H R V A D H A QV V R V L G F F Q C H S H P A Q A F D D A M T QF G M S R H G L L Q L F R R V G V T E L E A R SG T L P P A S Q R W D R I L Q A S G M K R A K PS P T S T Q T P D Q A S L H A F A D S L E R D LD A P S P M H E G D Q T R A S

As used herein the predetermined “N-terminus” to “C terminus”orientation of the N-terminal capping region, the DNA binding domaincomprising the repeat TALE monomers and the C-terminal capping regionprovide structural basis for the organization of different domains inthe d-TALEs or polypeptides of the invention.

The entire N-terminal and/or C-terminal capping regions are notnecessary to enhance the binding activity of the DNA binding region.Therefore, in certain embodiments, fragments of the N-terminal and/orC-terminal capping regions are included in the TALE polypeptidesdescribed herein.

In certain embodiments, the TALE polypeptides described herein contain aN-terminal capping region fragment that included at least 10, 20, 30,40, 50, 54, 60, 70, 80, 87, 90, 94, 100, 102, 110, 117, 120, 130, 140,147, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or 270amino acids of an N-terminal capping region. In certain embodiments, theN-terminal capping region fragment amino acids are of the C-terminus(the DNA-binding region proximal end) of an N-terminal capping region.As described in Zhang et al., Nature Biotechnology 29:149-153 (2011),N-terminal capping region fragments that include the C-terminal 240amino acids enhance binding activity equal to the full length cappingregion, while fragments that include the C-terminal 147 amino acidsretain greater than 80% of the efficacy of the full length cappingregion, and fragments that include the C-terminal 117 amino acids retaingreater than 50% of the activity of the full-length capping region.

In some embodiments, the TALE polypeptides described herein contain aC-terminal capping region fragment that included at least 6, 10, 20, 30,37, 40, 50, 60, 68, 70, 80, 90, 100, 110, 120, 127, 130, 140, 150, 155,160, 170, 180 amino acids of a C-terminal capping region. In certainembodiments, the C-terminal capping region fragment amino acids are ofthe N-terminus (the DNA-binding region proximal end) of a C-terminalcapping region. As described in Zhang et al., Nature Biotechnology29:149-153 (2011), C-terminal capping region fragments that include theC-terminal 68 amino acids enhance binding activity equal to thefull-length capping region, while fragments that include the C-terminal20 amino acids retain greater than 50% of the efficacy of thefull-length capping region.

In certain embodiments, the capping regions of the TALE polypeptidesdescribed herein do not need to have identical sequences to the cappingregion sequences provided herein. Thus, in some embodiments, the cappingregion of the TALE polypeptides described herein have sequences that areat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical or share identity to the capping region aminoacid sequences provided herein. Sequence identity is related to sequencehomology. Homology comparisons may be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesecommercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences. In some preferred embodiments, the capping region of the TALEpolypeptides described herein have sequences that are at least 95%identical or share identity to the capping region amino acid sequencesprovided herein.

Sequence homologies can be generated by any of a number of computerprograms known in the art, which include but are not limited to BLAST orFASTA. Suitable computer programs for carrying out alignments like theGCG Wisconsin Bestfit package may also be used. Once the software hasproduced an optimal alignment, it is possible to calculate % homology,preferably % sequence identity. The software typically does this as partof the sequence comparison and generates a numerical result.

In some embodiments described herein, the TALE polypeptides of theinvention include a nucleic acid binding domain linked to the one ormore effector domains. The terms “effector domain” or “regulatory andfunctional domain” refer to a polypeptide sequence that has an activityother than binding to the nucleic acid sequence recognized by thenucleic acid binding domain. By combining a nucleic acid binding domainwith one or more effector domains, the polypeptides of the invention maybe used to target the one or more functions or activities mediated bythe effector domain to a particular target DNA sequence to which thenucleic acid binding domain specifically binds.

In some embodiments of the TALE polypeptides described herein, theactivity mediated by the effector domain is a biological activity. Forexample, in some embodiments the effector domain is a transcriptionalinhibitor (i.e., a repressor domain), such as an mSin interaction domain(SID). SID4X domain or a Kruppel-associated box (KRAB) or fragments ofthe KRAB domain. In some embodiments the effector domain is an enhancerof transcription (i.e. an activation domain), such as the VP16, VP64 orp65 activation domain. In some embodiments, the nucleic acid binding islinked, for example, with an effector domain that includes but is notlimited to a transposase, integrase, recombinase, resolvase, invertase,protease, DNA methyltransferase, DNA demethylase, histone acetylase,histone deacetylase, nuclease, transcriptional repressor,transcriptional activator, transcription factor recruiting, proteinnuclear-localization signal or cellular uptake signal.

In some embodiments, the effector domain is a protein domain whichexhibits activities which include but are not limited to transposaseactivity, integrase activity, recombinase activity, resolvase activity,invertase activity, protease activity, DNA methyltransferase activity,DNA demethylase activity, histone acetylase activity, histonedeacetylase activity, nuclease activity, nuclear-localization signalingactivity, transcriptional repressor activity, transcriptional activatoractivity, transcription factor recruiting activity, or cellular uptakesignaling activity. Other preferred embodiments of the invention mayinclude any combination of the activities described herein.

Meganucleases

In some embodiments, a meganuclease or system thereof can be used tomodify a polynucleotide. Meganucleases, which are endodeoxyribonucleasescharacterized by a large recognition site (double-stranded DNA sequencesof 12 to 40 base pairs). Exemplary methods for using meganucleases canbe found in U.S. Pat. Nos. 8,163,514, 8,133,697, 8,021,867, 8,119,361,8,119,381, 8,124,369, and 8,129,134, which are specifically incorporatedby reference.

Sequences Related to Nucleus Targeting and Transportation

In some embodiments, one or more components (e.g., the Cas proteinand/or deaminase, Zn Finger protein, TALE, or meganuclease) in thecomposition for engineering cells may comprise one or more sequencesrelated to nucleus targeting and transportation. Such sequence mayfacilitate the one or more components in the composition for targeting asequence within a cell. In order to improve targeting of the CRISPR-Casprotein and/or the nucleotide deaminase protein or catalytic domainthereof used in the methods of the present disclosure to the nucleus, itmay be advantageous to provide one or both of these components with oneor more nuclear localization sequences (NLSs).

In some embodiments, the NLSs used in the context of the presentdisclosure are heterologous to the proteins. Non-limiting examples ofNLSs include an NLS sequence derived from: the NLS of the SV40 viruslarge T-antigen, having the amino acid sequence PKKKRKV (SEQ ID No. 10)or PKKKRKVEAS (SEQ ID No. 11); the NLS from nucleoplasmin (e.g., thenucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ IDNo. 12)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ IDNo. 13) or RQRRNELKRSP (SEQ ID No. 14); the hRNPA1 M9 NLS having thesequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID No. 15); thesequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID No. 16) ofthe IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID No.17) and PPKKARED (SEQ ID No. 18) of the myoma T protein; the sequencePQPKKKPL (SEQ ID No. 19) of human p53; the sequence SALIKKKKKMAP (SEQ IDNo. 20) of mouse c-abl IV; the sequences DRLRR (SEQ ID No. 21) andPKQKKRK (SEQ ID No. 22) of the influenza virus NS1; the sequenceRKLKKKIKKL (SEQ ID No. 23) of the Hepatitis virus delta antigen; thesequence REKKKFLKRR (SEQ ID No. 24) of the mouse Mx1 protein; thesequence KRKGDEVDGVDEVAKKKSKK (SEQ ID No. 25) of the humanpoly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ IDNo. 26) of the steroid hormone receptors (human) glucocorticoid. Ingeneral, the one or more NLSs are of sufficient strength to driveaccumulation of the DNA-targeting Cas protein in a detectable amount inthe nucleus of a eukaryotic cell. In general, strength of nuclearlocalization activity may derive from the number of NLSs in theCRISPR-Cas protein, the particular NLS(s) used, or a combination ofthese factors. Detection of accumulation in the nucleus may be performedby any suitable technique. For example, a detectable marker may be fusedto the nucleic acid-targeting protein, such that location within a cellmay be visualized, such as in combination with a means for detecting thelocation of the nucleus (e.g., a stain specific for the nucleus such asDAPI). Cell nuclei may also be isolated from cells, the contents ofwhich may then be analyzed by any suitable process for detectingprotein, such as immunohistochemistry, Western blot, or enzyme activityassay. Accumulation in the nucleus may also be determined indirectly,such as by an assay for the effect of nucleic acid-targeting complexformation (e.g., assay for deaminase activity) at the target sequence,or assay for altered gene expression activity affected by DNA-targetingcomplex formation and/or DNA-targeting), as compared to a control notexposed to the CRISPR-Cas protein and deaminase protein, or exposed to aCRISPR-Cas and/or deaminase protein lacking the one or more NLSs.

The CRISPR-Cas and/or nucleotide deaminase proteins may be provided with1 or more, such as with, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreheterologous NLSs. In some embodiments, the proteins comprises about ormore than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or nearthe amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more NLSs at or near the carboxy-terminus, or a combination ofthese (e.g., zero or at least one or more NLS at the amino-terminus andzero or at one or more NLS at the carboxy terminus). When more than oneNLS is present, each may be selected independently of the others, suchthat a single NLS may be present in more than one copy and/or incombination with one or more other NLSs present in one or more copies.In some embodiments, an NLS is considered near the N- or C-terminus whenthe nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 40, 50, or more amino acids along the polypeptide chain fromthe N- or C-terminus. In preferred embodiments of the CRISPR-Casproteins, an NLS attached to the C-terminal of the protein.

In certain embodiments, the CRISPR-Cas protein and the deaminase proteinare delivered to the cell or expressed within the cell as separateproteins. In these embodiments, each of the CRISPR-Cas and deaminaseprotein can be provided with one or more NLSs as described herein. Incertain embodiments, the CRISPR-Cas and deaminase proteins are deliveredto the cell or expressed with the cell as a fusion protein. In theseembodiments one or both of the CRISPR-Cas and deaminase protein isprovided with one or more NLSs. Where the nucleotide deaminase is fusedto an adaptor protein (such as MS2) as described above, the one or moreNLS can be provided on the adaptor protein, provided that this does notinterfere with aptamer binding. In particular embodiments, the one ormore NLS sequences may also function as linker sequences between thenucleotide deaminase and the CRISPR-Cas protein.

In certain embodiments, guides of the disclosure comprise specificbinding sites (e.g. aptamers) for adapter proteins, which may be linkedto or fused to an nucleotide deaminase or catalytic domain thereof. Whensuch a guide forms a CRISPR complex (e.g., CRISPR-Cas protein binding toguide and target) the adapter proteins bind and, the nucleotidedeaminase or catalytic domain thereof associated with the adapterprotein is positioned in a spatial orientation which is advantageous forthe attributed function to be effective.

The skilled person will understand that modifications to the guide whichallow for binding of the adapter+nucleotide deaminase, but not properpositioning of the adapter+nucleotide deaminase (e.g. due to sterichindrance within the three dimensional structure of the CRISPR complex)are modifications which are not intended. The one or more modified guidemay be modified at the tetra loop, the stem loop 1, stem loop 2, or stemloop 3, as described herein, preferably at either the tetra loop or stemloop 2, and in some cases at both the tetra loop and stem loop 2.

In some embodiments, a component (e.g., the dead Cas protein, thenucleotide deaminase protein or catalytic domain thereof, or acombination thereof) in the systems may comprise one or more nuclearexport signals (NES), one or more nuclear localization signals (NLS), orany combinations thereof. In some cases, the NES may be an HIV Rev NES.In certain cases, the NES may be MAPK NES. When the component is aprotein, the NES or NLS may be at the C terminus of component.Alternatively or additionally, the NES or NLS may be at the N terminusof component. In some examples, the Cas protein and optionally saidnucleotide deaminase protein or catalytic domain thereof comprise one ormore heterologous nuclear export signal(s) (NES(s)) or nuclearlocalization signal(s) (NLS(s)), preferably an HIV Rev NES or MAPK NES,preferably C-terminal.

Templates

In some embodiments, the composition for engineering cells comprise atemplate, e.g., a recombination template. A template may be a componentof another vector as described herein, contained in a separate vector,or provided as a separate polynucleotide. In some embodiments, arecombination template is designed to serve as a template in homologousrecombination, such as within or near a target sequence nicked orcleaved by a nucleic acid-targeting effector protein as a part of anucleic acid-targeting complex.

In an embodiment, the template nucleic acid alters the sequence of thetarget position. In an embodiment, the template nucleic acid results inthe incorporation of a modified, or non-naturally occurring base intothe target nucleic acid.

The template sequence may undergo a breakage mediated or catalyzedrecombination with the target sequence. In an embodiment, the templatenucleic acid may include sequence that corresponds to a site on thetarget sequence that is cleaved by a Cas protein mediated cleavageevent. In an embodiment, the template nucleic acid may include sequencethat corresponds to both, a first site on the target sequence that iscleaved in a first Cas protein mediated event, and a second site on thetarget sequence that is cleaved in a second Cas protein mediated event.

In certain embodiments, the template nucleic acid can include sequencewhich results in an alteration in the coding sequence of a translatedsequence, e.g., one which results in the substitution of one amino acidfor another in a protein product, e.g., transforming a mutant alleleinto a wild type allele, transforming a wild type allele into a mutantallele, and/or introducing a stop codon, insertion of an amino acidresidue, deletion of an amino acid residue, or a nonsense mutation. Incertain embodiments, the template nucleic acid can include sequencewhich results in an alteration in a non-coding sequence, e.g., analteration in an exon or in a 5′ or 3′ non-translated or non-transcribedregion. Such alterations include an alteration in a control element,e.g., a promoter, enhancer, and an alteration in a cis-acting ortrans-acting control element.

A template nucleic acid having homology with a target position in atarget gene may be used to alter the structure of a target sequence. Thetemplate sequence may be used to alter an unwanted structure, e.g., anunwanted or mutant nucleotide. The template nucleic acid may includesequence which, when integrated, results in: decreasing the activity ofa positive control element; increasing the activity of a positivecontrol element; decreasing the activity of a negative control element;increasing the activity of a negative control element; decreasing theexpression of a gene; increasing the expression of a gene; increasingresistance to a disorder or disease; increasing resistance to viralentry; correcting a mutation or altering an unwanted amino acid residueconferring, increasing, abolishing or decreasing a biological propertyof a gene product, e.g., increasing the enzymatic activity of an enzyme,or increasing the ability of a gene product to interact with anothermolecule.

The template nucleic acid may include sequence which results in: achange in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12 or morenucleotides of the target sequence.

A template polynucleotide may be of any suitable length, such as aboutor more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, ormore nucleotides in length. In an embodiment, the template nucleic acidmay be 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10,90+/−10, 100+/−10, 1 10+/−10, 120+/−10, 130+/−10, 140+/−10, 150+/−10,160+/−10, 170+/−10, 1 80+/−10, 190+/−10, 200+/−10, 210+/−10, of 220+/−10nucleotides in length. In an embodiment, the template nucleic acid maybe 30+/−20, 40+/−20, 50+/−20, 60+/−20, 70+/−20, 80+/−20, 90+/−20,100+/−20, 1 10+/−20, 120+/−20, 130+/−20, 140+/−20, 150+/−20, 160+/−20,170+/−20, 180+/−20, 190+/−20, 200+/−20, 210+/−20, of 220+/−20nucleotides in length. In an embodiment, the template nucleic acid is 10to 1 ,000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 50 to 500, 50 to400, 50 to300, 50 to 200, or 50 to 100 nucleotides in length.

In some embodiments, the template polynucleotide is complementary to aportion of a polynucleotide comprising the target sequence. Whenoptimally aligned, a template polynucleotide might overlap with one ormore nucleotides of a target sequences (e.g. about or more than about 1,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or morenucleotides). In some embodiments, when a template sequence and apolynucleotide comprising a target sequence are optimally aligned, thenearest nucleotide of the template polynucleotide is within about 1, 5,10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, ormore nucleotides from the target sequence.

The exogenous polynucleotide template comprises a sequence to beintegrated (e.g., a mutated gene). The sequence for integration may be asequence endogenous or exogenous to the cell. Examples of a sequence tobe integrated include polynucleotides encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction.

An upstream or downstream sequence may comprise from about 20 bp toabout 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, the exemplaryupstream or downstream sequence have about 200 bp to about 2000 bp,about 600 bp to about 1000 bp, or more particularly about 700 bp toabout 1000.

An upstream or downstream sequence may comprise from about 20 bp toabout 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, the exemplaryupstream or downstream sequence have about 200 bp to about 2000 bp,about 600 bp to about 1000 bp, or more particularly about 700 bp toabout 1000

In certain embodiments, one or both homology arms may be shortened toavoid including certain sequence repeat elements. For example, a 5′homology arm may be shortened to avoid a sequence repeat element. Inother embodiments, a 3′ homology arm may be shortened to avoid asequence repeat element. In some embodiments, both the 5′ and the 3′homology arms may be shortened to avoid including certain sequencerepeat elements.

In some methods, the exogenous polynucleotide template may furthercomprise a marker. Such a marker may make it easy to screen for targetedintegrations. Examples of suitable markers include restriction sites,fluorescent proteins, or selectable markers. The exogenouspolynucleotide template of the disclosure can be constructed usingrecombinant techniques (see, for example, Sambrook et al., 2001 andAusubel et al., 1996).

In certain embodiments, a template nucleic acid for correcting amutation may be designed for use as a single-stranded oligonucleotide.When using a single-stranded oligonucleotide, 5′ and 3′ homology armsmay range up to about 200 base pairs (bp) in length, e.g., at least 25,50, 75, 100, 125, 150, 175, or 200 bp in length.

In certain embodiments, a template nucleic acid for correcting amutation may be designed for use with a homology-independent targetedintegration system. Suzuki et al. describe in vivo genome editing viaCRISPR/Cas9 mediated homology-independent targeted integration (2016,Nature 540:144-149). Schmid-Burgk, et al. describe use of theCRISPR-Cas9 system to introduce a double-strand break (DSB) at auser-defined genomic location and insertion of a universal donor DNA(Nat Commun. 2016 Jul 28;7:12338). Gao, et al. describe “Plug-and-PlayProtein Modification Using Homology-Independent Universal GenomeEngineering” (Neuron. 2019 Aug. 21; 103(4):583-597).

RNAi

In some embodiments, the genetic modulating agents may be interferingRNAs. In certain embodiments, diseases caused by a dominant mutation ina gene is targeted by silencing the mutated gene using RNAi. In somecases, the nucleotide sequence may comprise coding sequence for one ormore interfering RNAs. In certain examples, the nucleotide sequence maybe interfering RNA (RNAi). As used herein, the term “RNAi” refers to anytype of interfering RNA, including but not limited to, siRNAi, shRNAi,endogenous microRNA and artificial microRNA. For instance, it includessequences previously identified as siRNA, regardless of the mechanism ofdown-stream processing of the RNA (i.e. although siRNAs are believed tohave a specific method of in vivo processing resulting in the cleavageof mRNA, such sequences can be incorporated into the vectors in thecontext of the flanking sequences described herein). The term “RNAi” caninclude both gene silencing RNAi molecules, and also RNAi effectormolecules which activate the expression of a gene.

In certain embodiments, a modulating agent may comprise silencing one ormore endogenous genes. As used herein, “gene silencing” or “genesilenced” in reference to an activity of an RNAi molecule, for example asiRNA or miRNA refers to a decrease in the mRNA level in a cell for atarget gene by at least about 5%, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%,about 99%, about 100% of the mRNA level found in the cell without thepresence of the miRNA or RNA interference molecule. In one preferredembodiment, the mRNA levels are decreased by at least about 70%, about80%, about 90%, about 95%, about 99%, about 100%.

As used herein, a “siRNA” refers to a nucleic acid that forms a doublestranded RNA, which double stranded RNA has the ability to reduce orinhibit expression of a gene or target gene when the siRNA is present orexpressed in the same cell as the target gene. The double stranded RNAsiRNA can be formed by the complementary strands. In one embodiment, asiRNA refers to a nucleic acid that can form a double stranded siRNA.The sequence of the siRNA can correspond to the full-length target gene,or a subsequence thereof. Typically, the siRNA is at least about 15-50nucleotides in length (e.g., each complementary sequence of the doublestranded siRNA is about 15-50 nucleotides in length, and the doublestranded siRNA is about 15-50 base pairs in length, preferably about19-30 base nucleotides, preferably about 20-25 nucleotides in length,e.g., 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides inlength).

As used herein “shRNA” or “small hairpin RNA” (also called stem loop) isa type of siRNA. In one embodiment, these shRNAs are composed of ashort, e.g. about 19 to about 25 nucleotide, antisense strand, followedby a nucleotide loop of about 5 to about 9 nucleotides, and theanalogous sense strand. Alternatively, the sense strand can precede thenucleotide loop structure and the antisense strand can follow.

The terms “microRNA” or “miRNA” are used interchangeably herein areendogenous RNAs, some of which are known to regulate the expression ofprotein-coding genes at the posttranscriptional level. EndogenousmicroRNAs are small RNAs naturally present in the genome that arecapable of modulating the productive utilization of mRNA. The termartificial microRNA includes any type of RNA sequence, other thanendogenous microRNA, which is capable of modulating the productiveutilization of mRNA. MicroRNA sequences have been described inpublications such as Lim, et al., Genes & Development, 17, p. 991-1008(2003), Lim et al Science 299, 1540 (2003), Lee and Ambros Science, 294,862 (2001), Lau et al., Science 294, 858-861 (2001), Lagos-Quintana etal, Current Biology, 12, 735-739 (2002), Lagos Quintana et al, Science294, 853-857 (2001), and Lagos-Quintana et al, RNA, 9, 175-179 (2003),which are incorporated by reference. Multiple microRNAs can also beincorporated into a precursor molecule. Furthermore, miRNA-likestem-loops can be expressed in cells as a vehicle to deliver artificialmiRNAs and short interfering RNAs (siRNAs) for the purpose of modulatingthe expression of endogenous genes through the miRNA and or RNAipathways.

As used herein, “double stranded RNA” or “dsRNA” refers to RNA moleculesthat are comprised of two strands. Double-stranded molecules includethose comprised of a single RNA molecule that doubles back on itself toform a two-stranded structure. For example, the stem loop structure ofthe progenitor molecules from which the single-stranded miRNA isderived, called the pre-miRNA (Bartel et al. 2004. Cell 1 16:281-297),comprises a dsRNA molecule.

Detection of Biomarkers

In certain embodiments, the invention provides uses of the biomarkersfor predicting risk for a certain phenotype. In certain embodiments, theinvention provides uses of the biomarkers for selecting a treatment. Incertain embodiments, a subject having a disease can be classified basedon severity of the disease. In certain embodiments, a tumor is monitoredfor expression of a biomarker described herein.

The terms “diagnosis” and “monitoring” are commonplace andwell-understood in medical practice. By means of further explanation andwithout limitation the term “diagnosis” generally refers to the processor act of recognizing, deciding on or concluding on a disease orcondition in a subject on the basis of symptoms and signs and/or fromresults of various diagnostic procedures (such as, for example, fromknowing the presence, absence and/or quantity of one or more biomarkerscharacteristic of the diagnosed disease or condition).

The terms “prognosing” or “prognosis” generally refer to an anticipationon the progression of a disease or condition and the prospect (e.g., theprobability, duration, and/or extent) of recovery. A good prognosis ofthe diseases or conditions taught herein may generally encompassanticipation of a satisfactory partial or complete recovery from thediseases or conditions, preferably within an acceptable time period. Agood prognosis of such may more commonly encompass anticipation of notfurther worsening or aggravating of such, preferably within a given timeperiod. A poor prognosis of the diseases or conditions as taught hereinmay generally encompass anticipation of a substandard recovery and/orunsatisfactorily slow recovery, or to substantially no recovery or evenfurther worsening of such.

The biomarkers of the present invention are useful in methods ofidentifying specific patient populations based on a detected level ofexpression, activity and/or function of one or more biomarkers. Thesebiomarkers are also useful in monitoring subjects undergoing treatmentsand therapies for suitable or aberrant response(s) to determineefficaciousness of the treatment or therapy and for selecting ormodifying therapies and treatments that would be efficacious intreating, delaying the progression of or otherwise ameliorating asymptom. The biomarkers provided herein are useful for selecting a groupof patients at a specific state of a disease with accuracy thatfacilitates selection of treatments.

The term “monitoring” generally refers to the follow-up of a disease ora condition in a subject for any changes which may occur over time.

The terms also encompass prediction of a disease. The terms “predicting”or “prediction” generally refer to an advance declaration, indication orforetelling of a disease or condition in a subject not (yet) having saiddisease or condition. For example, a prediction of a disease orcondition in a subject may indicate a probability, chance or risk thatthe subject will develop said disease or condition, for example within acertain time period or by a certain age. Said probability, chance orrisk may be indicated inter alfa as an absolute value, range orstatistics, or may be indicated relative to a suitable control subjector subject population (such as, e.g., relative to a general, normal orhealthy subject or subject population). Hence, the probability, chanceor risk that a subject will develop a disease or condition may beadvantageously indicated as increased or decreased, or as fold-increasedor fold-decreased relative to a suitable control subject or subjectpopulation. As used herein, the term “prediction” of the conditions ordiseases as taught herein in a subject may also particularly mean thatthe subject has a ‘positive’ prediction of such, i.e., that the subjectis at risk of having such (e.g., the risk is significantly increasedvis-à-vis a control subject or subject population). The term “predictionof no” diseases or conditions as taught herein as described herein in asubject may particularly mean that the subject has a ‘negative’prediction of such, i.e., that the subject's risk of having such is notsignificantly increased vis-à-vis a control subject or subjectpopulation.

Hence, the methods may rely on comparing the quantity of biomarkers, orgene or gene product signatures measured in samples from patients withreference values, wherein said reference values represent knownpredictions, diagnoses and/or prognoses of diseases or conditions astaught herein.

For example, distinct reference values may represent the prediction of arisk (e.g., an abnormally elevated risk) of having a given disease orcondition as taught herein vs. the prediction of no or normal risk ofhaving said disease or condition. In another example, distinct referencevalues may represent predictions of differing degrees of risk of havingsuch disease or condition.

In a further example, distinct reference values can represent thediagnosis of a given disease or condition as taught herein vs. thediagnosis of no such disease or condition (such as, e.g., the diagnosisof healthy, or recovered from said disease or condition, etc.). Inanother example, distinct reference values may represent the diagnosisof such disease or condition of varying severity.

In yet another example, distinct reference values may represent a goodprognosis for a given disease or condition as taught herein vs. a poorprognosis for said disease or condition. In a further example, distinctreference values may represent varyingly favorable or unfavorableprognoses for such disease or condition.

Such comparison may generally include any means to determine thepresence or absence of at least one difference and optionally of thesize of such difference between values being compared. A comparison mayinclude a visual inspection, an arithmetical or statistical comparisonof measurements. Such statistical comparisons include, but are notlimited to, applying a rule.

Reference values may be established according to known procedurespreviously employed for other cell populations, biomarkers and gene orgene product signatures. For example, a reference value may beestablished in an individual or a population of individualscharacterized by a particular diagnosis, prediction and/or prognosis ofsaid disease or condition (i.e., for whom said diagnosis, predictionand/or prognosis of the disease or condition holds true). Suchpopulation may comprise without limitation 2 or more, 10 or more, 100 ormore, or even several hundred or more individuals.

A “deviation” of a first value from a second value may generallyencompass any direction (e.g., increase: first value>second value; ordecrease: first value<second value) and any extent of alteration.

For example, a deviation may encompass a decrease in a first value by,without limitation, at least about 10% (about 0.9-fold or less), or byat least about 20% (about 0.8-fold or less), or by at least about 30%(about 0.7-fold or less), or by at least about 40% (about 0.6-fold orless), or by at least about 50% (about 0.5-fold or less), or by at leastabout 60% (about 0.4-fold or less), or by at least about 70% (about0.3-fold or less), or by at least about 80% (about 0.2-fold or less), orby at least about 90% (about 0.1-fold or less), relative to a secondvalue with which a comparison is being made.

For example, a deviation may encompass an increase of a first value by,without limitation, at least about 10% (about 1.1-fold or more), or byat least about 20% (about 1.2-fold or more), or by at least about 30%(about 1.3-fold or more), or by at least about 40% (about 1.4-fold ormore), or by at least about 50% (about 1.5-fold or more), or by at leastabout 60% (about 1.6-fold or more), or by at least about 70% (about1.7-fold or more), or by at least about 80% (about 1.8-fold or more), orby at least about 90% (about 1.9-fold or more), or by at least about100% (about 2-fold or more), or by at least about 150% (about 2.5-foldor more), or by at least about 200% (about 3-fold or more), or by atleast about 500% (about 6-fold or more), or by at least about 700%(about 8-fold or more), or like, relative to a second value with which acomparison is being made.

Preferably, a deviation may refer to a statistically significantobserved alteration. For example, a deviation may refer to an observedalteration which falls outside of error margins of reference values in agiven population (as expressed, for example, by standard deviation orstandard error, or by a predetermined multiple thereof, e.g., ±1xSD or±2xSD or ±3xSD, or ±1xSE or ±2xSE or ±3xSE). Deviation may also refer toa value falling outside of a reference range defined by values in agiven population (for example, outside of a range which comprises ≥40%,≥50%, ≥60%, ≥70%, ≥75% or ≥80% or ≥85% or ≥90% or ≥95% or even ≥100% ofvalues in said population).

In a further embodiment, a deviation may be concluded if an observedalteration is beyond a given threshold or cut-off. Such threshold orcut-off may be selected as generally known in the art to provide for achosen sensitivity and/or specificity of the prediction methods, e.g.,sensitivity and/or specificity of at least 50%, or at least 60%, or atleast 70%, or at least 80%, or at least 85%, or at least 90%, or atleast 95%.

For example, receiver-operating characteristic (ROC) curve analysis canbe used to select an optimal cut-off value of the quantity of a givenimmune cell population, biomarker or gene or gene product signatures,for clinical use of the present diagnostic tests, based on acceptablesensitivity and specificity, or related performance measures which arewell-known per se, such as positive predictive value (PPV), negativepredictive value (NPV), positive likelihood ratio (LR+), negativelikelihood ratio (LR−), Youden index, or similar.

In one embodiment, the signature genes, biomarkers, and/or cellsexpressing biomarkers may be detected or isolated by immunofluorescence,immunohistochemistry (IHC), fluorescence activated cell sorting (FACS),mass spectrometry (MS), mass cytometry (CyTOF), sequencing, WGS(described herein), WES (described herein), RNA-seq, single cell RNA-seq(described herein), quantitative RT-PCR, single cell qPCR, FISH,RNA-FISH, MERFISH (multiplex (in situ) RNA FISH) and/or by in situhybridization. Other methods including absorbance assays andcolorimetric assays are known in the art and may be used herein.Detection may comprise primers and/or probes or fluorescently bar-codedoligonucleotide probes for hybridization to RNA (see e.g., Geiss G K, etal., Direct multiplexed measurement of gene expression with color-codedprobe pairs. Nat Biotechnol. 2008 March; 26(3):317-25). In certainembodiments, cancer is diagnosed, prognosed, or monitored. For example,a tissue sample may be obtained and analyzed for specific cell markers(IHC) or specific transcripts (e.g., RNA-FISH). In one embodiment, tumorcells are stained for cell subtype specific signature genes. In oneembodiment, the cells are fixed. In another embodiment, the cells areformalin fixed and paraffin embedded. Not being bound by a theory, thepresence of the tumor subtypes indicate outcome and personalizedtreatments.

The present invention also may comprise a kit with a detection reagentthat binds to one or more biomarkers or can be used to detect one ormore biomarkers.

MS Methods

Biomarker detection may also be evaluated using mass spectrometrymethods. A variety of configurations of mass spectrometers can be usedto detect biomarker values. Several types of mass spectrometers areavailable or can be produced with various configurations. In general, amass spectrometer has the following major components: a sample inlet, anion source, a mass analyzer, a detector, a vacuum system, andinstrument-control system, and a data system. Difference in the sampleinlet, ion source, and mass analyzer generally define the type ofinstrument and its capabilities. For example, an inlet can be acapillary-column liquid chromatography source or can be a direct probeor stage such as used in matrix-assisted laser desorption. Common ionsources are, for example, electrospray, including nanospray andmicrospray or matrix-assisted laser desorption. Common mass analyzersinclude a quadrupole mass filter, ion trap mass analyzer andtime-of-flight mass analyzer. Additional mass spectrometry methods arewell known in the art (see Burlingame et al., Anal. Chem. 70:647 R-716R(1998); Kinter and Sherman, New York (2000)).

Protein biomarkers and biomarker values can be detected and measured byany of the following: electrospray ionization mass spectrometry(ESI-MS), ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorptionionization time-of-flight mass spectrometry (MALDI-TOF-MS),surface-enhanced laser desorption/ionization time-of-flight massspectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS),secondary ion mass spectrometry (SIMS), quadrupole time-of-flight(Q-TOF), tandem time-of-flight (TOF/TOF) technology, called ultraflexIII TOF/TOF, atmospheric pressure chemical ionization mass spectrometry(APCI-MS), APCI-MS/MS, APCI-(MS).sup.N, atmospheric pressurephotoionization mass spectrometry (APPI-MS), APPI-MS/MS, andAPPI-(MS).sup.N, quadrupole mass spectrometry, Fourier transform massspectrometry (FTMS), quantitative mass spectrometry, and ion trap massspectrometry.

Sample preparation strategies are used to label and enrich samplesbefore mass spectroscopic characterization of protein biomarkers anddetermination biomarker values. Labeling methods include but are notlimited to isobaric tag for relative and absolute quantitation (iTRAQ)and stable isotope labeling with amino acids in cell culture (SILAC).Capture reagents used to selectively enrich samples for candidatebiomarker proteins prior to mass spectroscopic analysis include but arenot limited to aptamers, antibodies, nucleic acid probes, chimeras,small molecules, an F(ab′)₂ fragment, a single chain antibody fragment,an Fv fragment, a single chain Fv fragment, a nucleic acid, a lectin, aligand-binding receptor, affibodies, nanobodies, ankyrins, domainantibodies, alternative antibody scaffolds (e.g. diabodies etc.)imprinted polymers, avimers, peptidomimetics, peptoids, peptide nucleicacids, threose nucleic acid, a hormone receptor, a cytokine receptor,and synthetic receptors, and modifications and fragments of these.

Immunoassays

Immunoassay methods are based on the reaction of an antibody to itscorresponding target or analyte and can detect the analyte in a sampledepending on the specific assay format. To improve specificity andsensitivity of an assay method based on immunoreactivity, monoclonalantibodies are often used because of their specific epitope recognition.Polyclonal antibodies have also been successfully used in variousimmunoassays because of their increased affinity for the target ascompared to monoclonal antibodies Immunoassays have been designed foruse with a wide range of biological sample matrices Immunoassay formatshave been designed to provide qualitative, semi-quantitative, andquantitative results.

Quantitative results may be generated through the use of a standardcurve created with known concentrations of the specific analyte to bedetected. The response or signal from an unknown sample is plotted ontothe standard curve, and a quantity or value corresponding to the targetin the unknown sample is established.

Numerous immunoassay formats have been designed. ELISA or EIA can bequantitative for the detection of an analyte/biomarker. This methodrelies on attachment of a label to either the analyte or the antibodyand the label component includes, either directly or indirectly, anenzyme. ELISA tests may be formatted for direct, indirect, competitive,or sandwich detection of the analyte. Other methods rely on labels suchas, for example, radioisotopes (I¹²⁵) or fluorescence. Additionaltechniques include, for example, agglutination, nephelometry,turbidimetry, Western blot, immunoprecipitation, immunocytochemi stry,immunohistochemistry, flow cytometry, Luminex assay, and others (seeImmunoAssay: A Practical Guide, edited by Brian Law, published by Taylor& Francis, Ltd., 2005 edition).

Exemplary assay formats include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay, fluorescent, chemiluminescence, andfluorescence resonance energy transfer (FRET) or time resolved-FRET(TR-FRET) immunoassays. Examples of procedures for detecting biomarkersinclude biomarker immunoprecipitation followed by quantitative methodsthat allow size and peptide level discrimination, such as gelelectrophoresis, capillary electrophoresis, planarelectrochromatography, and the like.

Methods of detecting and/or quantifying a detectable label or signalgenerating material depend on the nature of the label. The products ofreactions catalyzed by appropriate enzymes (where the detectable labelis an enzyme; see above) can be, without limitation, fluorescent,luminescent, or radioactive or they may absorb visible or ultravioletlight. Examples of detectors suitable for detecting such detectablelabels include, without limitation, x-ray film, radioactivity counters,scintillation counters, spectrophotometers, colorimeters, fluorometers,luminometers, and densitometers.

Any of the methods for detection can be performed in any format thatallows for any suitable preparation, processing, and analysis of thereactions. This can be, for example, in multi-well assay plates (e.g.,96 wells or 384 wells) or using any suitable array or microarray. Stocksolutions for various agents can be made manually or robotically, andall subsequent pipetting, diluting, mixing, distribution, washing,incubating, sample readout, data collection and analysis can be donerobotically using commercially available analysis software, robotics,and detection instrumentation capable of detecting a detectable label.

Hybridization Assays

Such applications are hybridization assays in which a nucleic acid thatdisplays “probe” nucleic acids for each of the genes to beassayed/profiled in the profile to be generated is employed. In theseassays, a sample of target nucleic acids is first prepared from theinitial nucleic acid sample being assayed, where preparation may includelabeling of the target nucleic acids with a label, e.g., a member of asignal producing system. Following target nucleic acid samplepreparation, the sample is contacted with the array under hybridizationconditions, whereby complexes are formed between target nucleic acidsthat are complementary to probe sequences attached to the array surface.The presence of hybridized complexes is then detected, eitherqualitatively or quantitatively. Specific hybridization technology whichmay be practiced to generate the expression profiles employed in thesubject methods includes the technology described in U.S. Pat. Nos.5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806;5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028;5,800,992; the disclosures of which are herein incorporated byreference; as well as WO 95/21265; WO 96/31622; WO 97/10365; WO97/27317; EP 373 203; and EP 785 280. In these methods, an array of“probe” nucleic acids that includes a probe for each of the biomarkerswhose expression is being assayed is contacted with target nucleic acidsas described above. Contact is carried out under hybridizationconditions, e.g., stringent hybridization conditions as described above,and unbound nucleic acid is then removed. The resultant pattern ofhybridized nucleic acids provides information regarding expression foreach of the biomarkers that have been probed, where the expressioninformation is in terms of whether or not the gene is expressed and,typically, at what level, where the expression data, i.e., expressionprofile, may be both qualitative and quantitative.

Optimal hybridization conditions will depend on the length (e.g.,oligomer vs. polynucleotide greater than 200 bases) and type (e.g., RNA,DNA, PNA) of labeled probe and immobilized polynucleotide oroligonucleotide. General parameters for specific (i.e., stringent)hybridization conditions for nucleic acids are described in Sambrook etal., supra, and in Ausubel et al., “Current Protocols in MolecularBiology”, Greene Publishing and Wiley-interscience, NY (1987), which isincorporated in its entirety for all purposes. When the cDNA microarraysare used, typical hybridization conditions are hybridization in 5×SSCplus 0.2% SDS at 65C for 4 hours followed by washes at 25° C. in lowstringency wash buffer (1×SSC plus 0.2% SDS) followed by 10 minutes at25° C. in high stringency wash buffer (0.1SSC plus 0.2% SDS) (see Shenaet al., Proc. Natl. Acad. Sci. USA, Vol. 93, p. 10614 (1996)). Usefulhybridization conditions are also provided in, e.g., Tijessen,Hybridization With Nucleic Acid Probes“, Elsevier Science PublishersB.V. (1993) and Kricka, “Nonisotopic DNA Probe Techniques”, AcademicPress, San Diego, Calif. (1992).

In certain embodiments, a subject can be categorized based on signaturegenes or gene programs expressed by a tissue sample obtained from thesubject. In certain embodiments, the tissue sample is analyzed by bulksequencing. In certain embodiments, subtypes can be determined bydetermining the percentage of specific cell subtypes expressing theidentified interacting genetic variants in the sample that contribute tothe phenotype. In certain embodiments, gene expression associated withthe cells are determined from bulk sequencing reads by deconvolution ofthe sample. For example, deconvoluting bulk gene expression dataobtained from a tumor containing both malignant and non-malignant cellscan include defining the relative frequency of a set of cell types inthe tumor from the bulk gene expression data using cell type specificgene expression (e.g., cell types may be T cells, fibroblasts,macrophages, mast cells, B/plasma cells, endothelial cells, myocytes anddendritic cells); and defining a linear relationship between thefrequency of the non-malignant cell types and the expression of a set ofgenes, wherein the set of genes comprises genes highly expressed bymalignant cells and at most two non-malignant cell types, wherein theset of genes are derived from gene expression analysis of single cellsin the tumor or the same tumor type, and wherein the residual of thelinear relationship defines the malignant cell-specific (MCS) expressionprofile (see, e.g., WO 2018/191553; and Puram et al., Cell. 2017 Dec.14; 171(7):1611-1624.e24).

Screening for Modulating Agents

In certain embodiments, the invention provides for screening fortherapeutic agents capable of altering the tumor microenvironment. Incertain embodiments, agents capable of blocking or enhancing interactingcell types are screened. In certain embodiments, the method comprises:a) applying a candidate agent to a cell population comprisinginteracting cells; b) detecting modulation of one or more phenotypicaspects of the cell population by the candidate agent, therebyidentifying the agent. The phenotypic aspects of the cell populationthat is modulated may be a gene signature specific to a cell type orcell phenotype or phenotype specific to a population of cells (e.g., ananti-tumor immune phenotype). In certain embodiments, steps can includeadministering candidate modulating agents to cells, detecting identifiedcell (sub)populations for changes in signatures, or identifying relativechanges in cell (sub) populations which may comprise detecting relativeabundance of particular gene signatures.

Tumor Models

In certain embodiments, therapeutic agents are screened or validated ina tumor model. In certain embodiments, cell-cell interactions in a tumorare identified using single cell methods for one or more tumor samples.In certain embodiments, associations to tumor progression (e.g., timeand size) are identified using single cell methods for one or more tumorsamples. In certain embodiments, the tumor sample can be obtained from atumor animal model. In certain embodiments, the tumor sample can beobtained from a mouse tumor model. Non-limiting examples of tumor mousemodels include the CT26 colon carcinoma, MC38-Ova colon carcinoma andB16F10 melanoma models (see, e.g., Singer, M. et al. A Distinct GeneModule for Dysfunction Uncoupled from Activation in Tumor-Infiltrating TCells. Cell 171, 1221-1223 (2017); and Kurtulus, S. et al. CheckpointBlockade Immunotherapy Induces Dynamic Changes in PD-1(−)CD8(+)Tumor-Infiltrating T Cells. Immunity 50, 181-194 e186 (2019)).

In certain embodiments, the tumor sample can be obtained over a timecourse to capture interactions occurring at specific time points duringtumor progression (see, e.g., International Patent Application Nos.PCT/US2018/053791, PCT/US2018/061812). The time course can be from 0 to365 days after implantation of a tumor in the mouse model. Time pointscan be taken at any day within the time course. In preferredembodiments, the time course is for about 20 days and includes samplestaken at about 5, 10, 15 and 20 days (e.g., day 11, 13, 15, 17 and 18).

In certain embodiments, the tumor sample can be obtained from one ormore subjects suffering from cancer. The sample may be a fresh sample orfrozen sample. Samples can be obtained from a subject over the course oftreating the cancer. Samples can be obtained before and after treatment.

In certain embodiments, the methods described herein are applicable toany cancer type. In preferred embodiments, the cancer is melanoma. Thecancer may include, without limitation, liquid tumors such as leukemia(e.g., acute leukemia, acute lymphocytic leukemia, acute myelocyticleukemia, acute myeloblastic leukemia, acute promyelocytic leukemia,acute myelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin'sdisease, non-Hodgkin's disease), Waldenstrom' s macroglobulinemia, heavychain disease, or multiple myeloma.

The cancer may include, without limitation, solid tumors such assarcomas and carcinomas. Examples of solid tumors include, but are notlimited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, epithelialcarcinoma, bronchogenic carcinoma, hepatoma, colorectal cancer (e.g.,colon cancer, rectal cancer), anal cancer, pancreatic cancer (e.g.,pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors),breast cancer (e.g., ductal carcinoma, lobular carcinoma, inflammatorybreast cancer, clear cell carcinoma, mucinous carcinoma), ovariancarcinoma (e.g., ovarian epithelial carcinoma or surfaceepithelial-stromal tumour including serous tumour, endometrioid tumorand mucinous cystadenocarcinoma, sex-cord-stromal tumor), prostatecancer, liver and bile duct carcinoma (e.g., hepatocelluar carcinoma,cholangiocarcinoma, hemangioma), choriocarcinoma, seminoma, embryonalcarcinoma, kidney cancer (e.g., renal cell carcinoma, clear cellcarcinoma, Wilm's tumor, nephroblastoma), cervical cancer, uterinecancer (e.g., endometrial adenocarcinoma, uterine papillary serouscarcinoma, uterine clear-cell carcinoma, uterine sarcomas andleiomyosarcomas, mixed mullerian tumors), testicular cancer, germ celltumor, lung cancer (e.g., lung adenocarcinoma, squamous cell carcinoma,large cell carcinoma, bronchioloalveolar carcinoma, non-small-cellcarcinoma, small cell carcinoma, mesothelioma), bladder carcinoma,signet ring cell carcinoma, cancer of the head and neck (e.g., squamouscell carcinomas), esophageal carcinoma (e.g., esophagealadenocarcinoma), tumors of the brain (e.g., glioma, glioblastoma,medullablastoma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodenroglioma, schwannoma, meningioma), neuroblastoma,retinoblastoma, neuroendocrine tumor, melanoma, cancer of the stomach(e.g., stomach adenocarcinoma, gastrointestinal stromal tumor), orcarcinoids. Lymphoproliferative disorders are also considered to beproliferative diseases.

Sequencing

In certain embodiments, sequencing is used to identify expression ofgenes or transcriptomes in single cells. In certain embodiments,sequencing comprises high-throughput (formerly “next-generation”)technologies to generate sequencing reads. Methods for constructingsequencing libraries are known in the art (see, e.g., Head et al.,Library construction for next-generation sequencing: Overviews andchallenges. Biotechniques. 2014; 56(2): 61-77). A “library” or “fragmentlibrary” may be a collection of nucleic acid molecules derived from oneor more nucleic acid samples, in which fragments of nucleic acid havebeen modified, generally by incorporating terminal adapter sequencescomprising one or more primer binding sites and identifiable sequencetags. In certain embodiments, the library members (e.g., cDNA) mayinclude sequencing adaptors that are compatible with use in, e.g.,Illumina's reversible terminator method, long read nanopore sequencing,Roche's pyrosequencing method (454), Life Technologies' sequencing byligation (the SOLiD platform) or Life Technologies' Ion Torrentplatform. Examples of such methods are described in the followingreferences: Margulies et al (Nature 2005 437: 376-80); Schneider andDekker (Nat Biotechnol. 2012 Apr 10;30(4):326-8); Ronaghi et al(Analytical Biochemistry 1996 242: 84-9); Shendure et al (Science 2005309: 1728-32); Imelfort et al (Brief Bioinform. 2009 10:609-18); Fox etal (Methods Mol. Biol. 2009; 553:79-108); Appleby et al (Methods Mol.Biol. 2009; 513:19-39); and Morozova et al (Genomics. 2008 92:255-64),which are incorporated by reference for the general descriptions of themethods and the particular steps of the methods, including all startingproducts, reagents, and final products for each of the steps.

As used herein the term “transcriptome” refers to the set of transcriptmolecules. In some embodiments, transcript refers to RNA molecules,e.g., messenger RNA (mRNA) molecules, small interfering RNA (siRNA)molecules, transfer RNA (tRNA) molecules, ribosomal RNA (rRNA)molecules, and complimentary sequences, e.g., cDNA molecules. In someembodiments, a transcriptome refers to a set of mRNA molecules. In someembodiments, a transcriptome refers to a set of cDNA molecules. In someembodiments, a transcriptome refers to one or more of mRNA molecules,siRNA molecules, tRNA molecules, rRNA molecules, in a sample, forexample, a single cell or a population of cells. In some embodiments, atranscriptome refers to cDNA generated from one or more of mRNAmolecules, siRNA molecules, tRNA molecules, rRNA molecules, in a sample,for example, a single cell or a population of cells. In someembodiments, a transcriptome refers to 25%, 50%, 55, 60, 65, 70, 75, 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9, or 100% of transcriptsfrom a single cell or a population of cells. In some embodiments,transcriptome not only refers to the species of transcripts, such asmRNA species, but also the amount of each species in the sample. In someembodiments, a transcriptome includes each mRNA molecule in the sample,such as all the mRNA molecules in a single cell.

In certain embodiments, the invention involves single cell RNAsequencing (see, e.g., Kalisky, T., Blainey, P. & Quake, S. R. GenomicAnalysis at the Single-Cell Level. Annual review of genetics 45,431-445, (2011); Kalisky, T. & Quake, S. R. Single-cell genomics. NatureMethods 8, 311-314 (2011); Islam, S. et al. Characterization of thesingle-cell transcriptional landscape by highly multiplex RNA-seq.Genome Research, (2011); Tang, F. et al. RNA-Seq analysis to capture thetranscriptome landscape of a single cell. Nature Protocols 5, 516-535,(2010); Tang, F. et al. mRNA-Seq whole-transcriptome analysis of asingle cell. Nature Methods 6, 377-382, (2009); Ramskold, D. et al.Full-length mRNA-Seq from single-cell levels of RNA and individualcirculating tumor cells. Nature Biotechnology 30, 777-782, (2012); andHashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-Seq: Single-CellRNA-Seq by Multiplexed Linear Amplification. Cell Reports, Cell Reports,Volume 2, Issue 3, p666-6′73, 2012).

In certain embodiments, the present invention involves single cell RNAsequencing (scRNA-seq). In certain embodiments, the invention involvesplate based single cell RNA sequencing (see, e.g., Picelli, S. et al.,2014, “Full-length RNA-seq from single cells using Smart-seq2” Natureprotocols 9, 171-181, doi:10.1038/nprot.2014.006).

In certain embodiments, the invention involves high-throughputsingle-cell RNA-seq where the RNAs from different cells are taggedindividually, allowing a single library to be created while retainingthe cell identity of each read. In this regard reference is made toMacosko et al., 2015, “Highly Parallel Genome-wide Expression Profilingof Individual Cells Using Nanoliter Droplets” Cell 161, 1202-1214;International patent application number PCT/US2015/049178, published asWO2016/040476 on Mar. 17, 2016; Klein et al., 2015, “Droplet Barcodingfor Single-Cell Transcriptomics Applied to Embryonic Stem Cells” Cell161, 1187-1201; International patent application numberPCT/US2016/027734, published as WO2016168584A1 on October 20, 2016;Zheng, et al., 2016, “Haplotyping germline and cancer genomes withhigh-throughput linked-read sequencing” Nature Biotechnology 34,303-311; Zheng, et al., 2017, “Massively parallel digitaltranscriptional profiling of single cells” Nat. Commun. 8, 14049 doi:10.1038/ncomms14049; International patent publication numberWO2014210353A2; Zilionis, et al., 2017, “Single-cell barcoding andsequencing using droplet microfluidics” Nat Protoc. Jan;12(1):44-73; Caoet al., 2017, “Comprehensive single cell transcriptional profiling of amulticellular organism by combinatorial indexing” bioRxiv preprint firstposted online Feb. 2, 2017, doi: dx.doi.org/10.1101/104844; Rosenberg etal., 2017, “Scaling single cell transcriptomics through split poolbarcoding” bioRxiv preprint first posted online Feb. 2, 2017, doi:dx.doi.org/10.1101/105163; Rosenberg et al., “Single-cell profiling ofthe developing mouse brain and spinal cord with split-pool barcoding”Science 15 Mar. 2018; Vitak, et al., “Sequencing thousands ofsingle-cell genomes with combinatorial indexing” Nature Methods,14(3):302-308, 2017; Cao, et al., Comprehensive single-celltranscriptional profiling of a multicellular organism. Science,357(6352):661-667, 2017; Gierahn et al., “Seq-Well: portable, low-costRNA sequencing of single cells at high throughput” Nature Methods 14,395-398 (2017); and Hughes, et al., “Highly Efficient,Massively-Parallel Single-Cell RNA-Seq Reveals Cellular States andMolecular Features of Human Skin Pathology” bioRxiv 689273; doi:doi.org/10.1101/689273, all the contents and disclosure of each of whichare herein incorporated by reference in their entirety.

In certain embodiments, the invention involves single nucleus RNAsequencing. In this regard reference is made to Swiech et al., 2014, “Invivo interrogation of gene function in the mammalian brain usingCRISPR-Cas9” Nature Biotechnology Vol. 33, pp. 102-106; Habib et al.,2016, “Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adultnewborn neurons” Science, Vol. 353, Issue 6302, pp. 925-928; Habib etal., 2017, “Massively parallel single-nucleus RNA-seq with DroNc-seq”Nat Methods. 2017 October; 14(10):955-958; International PatentApplication No. PCT/US2016/059239, published as WO2017164936 on Sep. 28,2017; International Patent Application No.PCT/US2018/060860, publishedas WO/2019/094984 on May 16, 2019; International Patent Application No.PCT/US2019/055894, published as WO/2020/077236 on Apr. 16, 2020; andDrokhlyansky, et al., “The enteric nervous system of the human and mousecolon at a single-cell resolution,” bioRxiv 746743; doi:doi.org/10.1101/746743, which are herein incorporated by reference intheir entirety.

Agents for Screening

In certain embodiments, agents are screened for inducing a phenotype.The term “agent” broadly encompasses any condition, substance or agentcapable of modulating one or more phenotypic aspects of a cell or cellpopulation as disclosed herein. Such conditions, substances or agentsmay be of physical, chemical, biochemical and/or biological nature. Theterm “candidate agent” refers to any condition, substance or agent thatis being examined for the ability to modulate one or more phenotypicaspects of a cell or cell population as disclosed herein in a methodcomprising applying the candidate agent to the cell or cell population(e.g., exposing the cell or cell population to the candidate agent orcontacting the cell or cell population with the candidate agent) andobserving whether the desired modulation takes place.

Agents may include any potential class of biologically activeconditions, substances or agents, such as for instance antibodies,proteins, peptides, nucleic acids, oligonucleotides, small molecules, orcombinations thereof, as described herein.

The methods of phenotypic analysis can be utilized for evaluatingenvironmental stress and/or state, for screening of chemical libraries,and to screen or identify structural, syntenic, genomic, and/or organismand species variations. For example, a culture of cells, can be exposedto an environmental stress, such as but not limited to heat shock,osmolarity, hypoxia, cold, oxidative stress, radiation, starvation, achemical (for example a therapeutic agent or potential therapeuticagent) and the like. After the stress is applied, a representativesample can be subjected to analysis, for example at various time points,and compared to a control, such as a sample from an organism or cell,for example a cell from an organism, or a standard value. By exposingcells, or fractions thereof, tissues, or even whole animals, todifferent members of the chemical libraries, and performing the methodsdescribed herein, different members of a chemical library can bescreened for their effect on immune phenotypes thereof simultaneously ina relatively short amount of time, for example using a high throughputmethod.

Aspects of the present disclosure relate to the correlation of an agentwith the spatial proximity and/or epigenetic profile of the nucleicacids in a sample of cells. In some embodiments, the disclosed methodscan be used to screen chemical libraries for agents that modulatechromatin architecture epigenetic profiles, and/or relationshipsthereof.

In some embodiments, screening of test agents involves testing acombinatorial library containing a large number of potential modulatorcompounds. A combinatorial chemical library may be a collection ofdiverse chemical compounds generated by either chemical synthesis orbiological synthesis, by combining a number of chemical “buildingblocks” such as reagents. For example, a linear combinatorial chemicallibrary, such as a polypeptide library, is formed by combining a set ofchemical building blocks (amino acids) in every possible way for a givencompound length (for example the number of amino acids in a polypeptidecompound). Millions of chemical compounds can be synthesized throughsuch combinatorial mixing of chemical building blocks.

In certain embodiments, the present invention provides for genesignature screening. The concept of signature screening was introducedby Stegmaier et al. (Gene expression-based high-throughput screening(GE-HTS) and application to leukemia differentiation. Nature Genet. 36,257-263 (2004)), who realized that if a gene-expression signature wasthe proxy for a phenotype of interest, it could be used to find smallmolecules that effect that phenotype without knowledge of a validateddrug target. The signatures or biological programs of the presentinvention may be used to screen for drugs that reduce the signature orbiological program in cells as described herein. The signature orbiological program may be used for GE-HTS. In certain embodiments,pharmacological screens may be used to identify drugs that areselectively toxic to cells having a signature.

The Connectivity Map (cmap) is a collection of genome-widetranscriptional expression data from cultured human cells treated withbioactive small molecules and simple pattern-matching algorithms thattogether enable the discovery of functional connections between drugs,genes and diseases through the transitory feature of commongene-expression changes (see, Lamb et al., The Connectivity Map: UsingGene-Expression Signatures to Connect Small Molecules, Genes, andDisease. Science 29 Sep. 2006: Vol. 313, Issue 5795, pp. 1929-1935, DOI:10.1126/science.1132939; and Lamb, J., The Connectivity Map: a new toolfor biomedical research. Nature Reviews Cancer January 2007: Vol. 7, pp.54-60). In certain embodiments, Cmap can be used to screen for smallmolecules capable of modulating a signature or biological program of thepresent invention in silico.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1 Single Cell Analysis of the Tumor MicroenvironmentOver Time

Applicants performed a time course study using B16 melanoma tumor mousemodels. Applicants performed single cell sequencing on the tumor cellsusing the 10× genomics platform. The single cell transcriptomes wereused to cluster the cell types and identify the cell types of eachcluster (FIG. 1). Applicants used 18 mice that passed quality controlsand cells were collected at different time points for single cellRNA-seq (FIG. 21-22).

Applicants further refined the cell clusters to an analysis of T celland non-T cell clusters and removed malignant cells from the analysis.The refined analysis was more extensive and annotates each cluster bycell type (FIGS. 23A and 25). UMAP analysis was performed on T cells andnon-T cells to identify clusters of cells. The cells were annotated bycell type using cell type marker genes (FIG. 23-26).

Example 2 Identification of Interacting Cells in the TumorMicroenvironment

Applicants identified interactions for clusters based on ligand-receptorinteractions under conditions where the ligands or receptors werespecific or generally upregulated for the gene clusters (FIGS. 2, 3, 4,5).

Applicants show that PDCD1 is expressed on CD8 T cells and its ligandPDCD1LG2 is expressed in clusters (FIG. 6A).

Applicants show that VEGFB is expressed exclusively by tumor cells andits ligand NRP1 is expressed on CD8 T cells (FIG. 6B). In endothelialcells, VEGF-B stimulation upregulated the expression of the fatty acid(FA) transport proteins (FATPs). In vivo, Hagberg et al. found thatVEGF-B deficient mice had a reduced FA uptake, leading to significantlyless FA accumulation in their hearts, muscles and brown adipose tissues(BATs) (Nature. 2010 Apr. 8; 464(7290):917-21). FA are preferred bymemory CD8 rather than effector CD8 T cells, which tend to rely more onglycolysis. Also, iTregs rely on FA metabolism and Foxp3 is a driver offatty acid oxidation enzymes expression. Thus, FA uptake by increasedexpression of VEGFB by tumors may increase Tregs in the tumormicroenvironment. Yang et al., show in a human melanoma xenograft modelthat VEGFB remodels microvasculature rendering it leaky and promotingcancer cell extravasation. Importantly, VEGF-B expression correlateswith poor prognosis (Proc Natl Acad Sci U S A. 2015 Jun. 2;112(22):E2900-9). Amongst the different human tumors (TCGA), VEGFBexpression is highest in melanoma (FIG. 19).

Applicants show that CCR1 is expressed exclusively by macrophages andits ligand CCL3 is expressed on CD8 T cells (FIG. 6C).

Applicants show that FBLN1 is expressed exclusively by tumor cells andits ligand ITGB1 is expressed on CD8 T cells (FIG. 20A). Thisinteraction does not seem to be direct, but through FN1 (FIG. 20C).FBLN1 expression along with FN1 has been implicated in breast cancerDoxorubicin resistance. In bladder cancer and colorectal cancer, FBLN1seems to correlate with a good prognosis. The role of FBLN1 in cancer isnot clear. FBLN1 may play a role in TGFβ activation along with FN1.

Applicants identified specific and generally interacting clusters (FIGS.7, 8). Applicants generated network interaction maps for all of theidentified clusters (FIGS. 9, 10). Applicants identified ligand receptornetworks and specific genes that are at the hubs of the networks (FIG.11A,B).

Integrin Alpha V (ITGAV) was found at one hub (FIG. 12). ITGAV is mainlyexpressed by Tregs. It encodes for integrin αV β1 subunit whichheterodimerizes with different β chains (αVβ1, αVβ3, αVβ5, αVβ6 andαVβ8). This integrin subfamily recognizes ligands containing theArg-Gly-Asp (RGD) tripeptide motif. Extracellular matrix (ECM)components, through specific peptide motifs such as Arg-Gly-Asp (RGD),interact with integrins and can modify the behavior of cells.Transforming growth factor-betal (TGF-betal) is the main cytokineinvolved in the synthesis of ECM proteins.

SDC4 was found at one hub (FIG. 13). Syndecan 4 (SDC4) is atransmembrane (type I) heparin sulfate proteoglycan. SDC4 can act as areceptor and co-receptor in mediating intracellular signaling. SDC4 isassociated with adhesion, membrane trafficking and has a role in MAPKand Mtor pathways.

Applicants also identified that the ITGAV network interacts with theSDC4 network through FN1 and TGM2 (FIG. 14). Applicants performedimmunohistochemistry to validate the interactions between cellsexpressing SDC4 (FIG. 16, 17), and Tregs expressing FN1 (ITGAV hub)(FIG. 18).

Applicants identified immune cells that interact directly with tumorcells. The tumor cells may suppress effector T cells and activate Tregsthrough interactions with the immune cells. Applicants identifiedinteractions between immune cells in the tumor microenvironment.Modulation of these interactions may also enhance an anti-tumorresponse. For example, immune cells may communicate with one another topromote the wound healing function, such as, throughITGAV-FN1-ITGBA-TGM2-SDC4 interactions. The interactions can be blockedin vivo to treat cancer. Applicants can generate effector T cells foradoptive cell transfer (CAR T, TILs) that are modified, such that theydo not interact with the tumor cells. In conclusion, the TME is anintricate milieu of cells hosting the tumor, including infiltratingmyeloid and lymphoid cells, stromal and mesenchymal cells, and ECMcomponents. Matrix remodeling shapes the inflamed immunemicroenvironment. Tumor-infiltrating Tregs and regulatory myeloid cells,including MDSCs, TAMs and TANs, promote a tolerogenic TME (FIG. 15). Theligand-receptor network data provide herein suggests that Treg cellsrepresent a central hub interacting with myeloid cells and activated CD8T cells, while orchestrating ECM remodeling.

Applicants used the cell cluster annotations (FIGS. 23A and 25) andsingle cell RNA expression data to identify up-regulated ligand-receptorpairs in the clusters and to characterize interactions amongst celltypes. Table 1 shows all of the ligand receptor pairs and the cell typesexpressing the ligand and receptor.

Applicants determined the cells having the most potential interactionsby quantitating the number of upregulated ligands or receptors for eachcluster (FIG. 27). Gene Set Enrichment Analysis (GSEA) was used toidentify statistically enriched ligand/receptor upregulation. Thenormalized enrichment score (NES) indicates clusters enriched inupregulated genes (above the line) (FIG. 28). Applicants analyzedcluster similarity (FIG. 29). Applicants generated network maps ofinteracting clusters (FIG. 30). The network edge equals an interactionand the width of the edge is the number of interactions. The wedgedensity is highest between T and non-T cells. Tregs have the highestinteractions between cells. Applicants determined gene networks forinteractions between ligand receptor pairs that are upregulated in theclusters (FIG. 31) and further determined gene networks for interactionsbetween T and non-T cell clusters (FIG. 32).

Various modifications and variations of the described methods,pharmaceutical compositions, and kits of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific embodiments, it will be understood that it iscapable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention. This application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure come within known customarypractice within the art to which the invention pertains and may beapplied to the essential features herein before set forth.

1. An isolated CD8 T cell modified to decrease or eliminate expression,activity or function of NRP1 or CRTAM.
 2. (canceled)
 3. The isolated CD8T cell of claim 1, wherein the T cells express a chimeric antigenreceptor (CAR) or endogenous T cell receptor (TCR); and/or wherein the Tcells are tumor infiltrating lymphocytes (TILs).
 4. (canceled)
 5. Amethod of enhancing an anti-tumor immune response in a subject in needthereof comprising: administering to the subject an antagonist ofVEGFB-NRP1 interaction, preferably, wherein the antagonist is anantibody or fragment thereof specific for NRP1 or VEGFB, oradministering to the subject an antagonist of CADM1-CRTAM interaction,preferably, wherein the antagonist is an antibody or fragment thereofspecific for CADM1 or CRTAM, or administering to the subject anantagonist of TFPI-SDC4 interaction, preferably, wherein the antagonistis an antibody or fragment thereof specific for TFPI or SDC4, oradministering to the subject an antagonist of MFGE8-ITGAV interaction,preferably, wherein the antagonist is an antibody or fragment thereofspecific for MFGE8 or ITGAV, or administering to the subject anantagonist of ADAM12-SDC4 interaction, preferably, wherein theantagonist is an antibody or fragment thereof specific for ADAM12 orSDC4, or administering to the subject an antagonist of CYR61-ITGAVinteraction, preferably, wherein the antagonist is an antibody orfragment thereof specific for CYR61 or ITGAV, or administering to thesubject an antagonist of the interaction between FN1 with FBLN1 and/orITGB1, preferably, wherein the antagonist is an antibody or fragmentthereof specific for FN1, FBLN1, or ITGB1; or administering to thesubject an antagonist of one or more ligand-receptor pairs in Table 1.6-27. (canceled)
 28. The method of claim 5, wherein the antagonist is asmall molecule, aptamer, or protein capable of binding to a receptor orligand.
 29. The method of claim 5, wherein the antagonist isadministered directly to the tumor, or wherein the antagonist isadministered intravenously or intraperitoneally to the subject; and/orwherein the antagonist is administered in a successive, sequential orsimultaneous treatment regimen; and/or wherein the subject isadministered an additional immunotherapy, preferably, wherein theimmunotherapy comprises anti-PD-1, anti-CTLA4, anti-PD-L1, anti-TIM3,anti-TIGIT, anti-LAG3, or combinations thereof. 30-33. (canceled)
 34. Amethod of generating T cells for adoptive cell transfer to a subjectsuffering from cancer comprising: a. obtaining T cells; and b. modifyingthe T cells to decrease or eliminate expression, activity or function ofone or more genes selected from the group consisting of NRP1 and CRTAM.35. The method of claim 34, wherein the T cells are modified with agenetic modifying agent capable of targeting the gene or genetranscript, preferably, wherein the genetic modifying agent comprises aCRISPR system, RNAi system, a zinc finger nuclease system, a TALEsystem, or a meganuclease, more preferably, wherein the CRISPR systemcomprises a CRISPR-Cas base editing system, a prime editor system, or aCAST system. 36-37. (canceled)
 38. The method of claim 34, wherein the Tcells are CD8 T cells; and/or wherein the T cells are further modifiedto express a chimeric antigen receptor (CAR) or exogenous T cellreceptor (TCR); and/or wherein the T cells are obtained from a subjectin need thereof, preferably, wherein the T cells are tumor infiltratinglymphocytes (TILS); and/or further comprising eliminating expression,activity or function of one or more additional genes selected from thegroup consisting of TRBC1, TRBC2 and TRAC; and/or further comprisingeliminating expression, activity or function of one or more additionalgenes selected from the group consisting of checkpoint proteins; and/orfurther comprising eliminating expression, activity or function of oneor more additional genes selected from the group consisting of HLA-A,HLA-B, HLA-C, and B2M. 39-41. (canceled)
 42. The method of claim 34,wherein the method comprises decreasing or eliminating expression,activity or function of NRP1.
 43. The method of claim 34, wherein themethod comprises decreasing or eliminating expression, activity orfunction of CRTAM. 44-46. (canceled)
 47. A method of cancer treatmentcomprising administering to a subject in need thereof the T cellsaccording to claim
 1. 48. A method of determining tumors resistant to animmune response comprising detecting the expression of a gene selectedfrom the group consisting of CADM1, VEGFB, TFPI, MFGE8, ADAM12, andFBLN1 in tumor cells obtained from a subject in need thereof, whereinincreased levels indicate resistant tumors. 49-53. (canceled)
 54. Themethod of claim 5, wherein the interaction between cell types disruptsthe extracellular matrix (ECM) in the tumor microenvironment andinitiates wound healing signaling.
 55. The method of claim 54, whereinthe agent is capable of blocking or disrupting wound healing signalingto Tregs.
 56. The method of claim 5, wherein the cancer is selected fromthe group consisting of melanoma, renal cancer, glioma, thyroid cancer,lung cancer, liver cancer, pancreatic cancer, head and neck cancer,stomach cancer, colorectal cancer, urothelial cancer, prostate cancer,testicular cancer, breast cancer, cervical cancer, ovarian cancer andendometrial cancer.
 57. A method of cancer treatment comprisingadministering to a subject in need thereof the T cells generatedaccording to claim 34.